Bacterial host strain comprising a mutant spr gene and a wild-type tsp gene

ABSTRACT

The present invention provides a recombinant gram-negative bacterial cell comprising a mutant spr gene encoding a mutant spr protein and wherein the cell comprises a non-recombinant wild-type chromosomal Tsp gene.

The invention relates to a recombinant bacterial host strain,particularly E. coli. The invention also relates to a method forproducing a protein of interest in such a cell.

BACKGROUND OF THE INVENTION

Bacterial cells, such as E. coli, are commonly used for producingrecombinant proteins. There are many advantages to using bacterialcells, such as E. coli, for producing recombinant proteins particularlydue to the versatile nature of bacterial cells as host cells allowingthe gene insertion via plasmids. E. coli have been used to produce manyrecombinant proteins including human insulin.

Despite the many advantages to using bacterial cells to producerecombinant proteins, there are still significant limitations includingthe tendency of bacterial cells to lyse during expression of arecombinant protein of interest. This lysis phenotype may be seen inwild-type bacterial cells and also genetically engineered cell, such ascells which are deficient in bacterial proteases. Proteases play animportant role in turning over old, damaged or miss-folded proteins inthe E. coli periplasm and cytoplasm. Bacterial proteases act to degradethe recombinant protein of interest, thereby often significantlyreducing the yield of active protein. Therefore, the reduction ofprotease activity is desirable to reduce proteolysis of proteins ofinterest. However, bacterial strains lacking proteases, such as Tsp(also known as Prc), also exhibit cell lysis.

Tsp (also known as Prc) is a 60 kDa periplasmic protease. The reductionof Tsp (prc) activity is desirable to reduce the proteolysis of proteinsof interest. However, it was found that cells lacking the protease prcshow thermosensitive growth at low osmolarity. Hara et al isolated Tspdeficient strains which were thermoresistant revertants containingextragenic suppressor (spr) mutations (Hara et al., Microbial DrugResistance, 2: 63-72 (1996)). Spr is an 18 kDa membrane boundperiplasmic protease and the substrates of spr are Tsp andpeptidoglycans in the outer membrane involved in cell wall hydrolysisduring cell division. The spr gene is designated as UniProtKB/Swiss-ProtPOAFV4 (SPR_ECOLI). Protease deficient bacterial strains carrying amutant spr gene have been described in Chen et al (Chen C, Snedecor B,Nishihara J C, Joly J C, McFarland N, Andersen D C, Battersby J E,Champion K M. Biotechnol Bioeng. 2004 Mar. 5; 85(5):463-74) whichdescribes the construction of E. coli strains carrying differentcombinations of mutations in prc (Tsp) and another protease, DegP,created by amplifying the upstream and downstream regions of the geneand ligating these together on a vector comprising selection markers anda sprW174R mutation

It has been surprisingly found that a gram-negative bacterial cellcarrying a mutant spr gene and a wild-type Tsp gene provides a cellhaving reduced lysis. Accordingly, the present inventors have provided anew strain having advantageous properties for producing a protein ofinterest.

It was surprising that cells according to the present invention showadvantageous growth and protein yield phenotype because spr and Tsp areknown to be mutual suppressors and, therefore, it would be predictedthat if one is allowed to dominate the cell may exhibit a poor growthphenotype, such as becoming leaky or show increase propensity to celllysis. However, the cells of the present invention exhibited asignificant reduction in cell lysis phenotype compared to wild-typecells and cells comprising a knockout mutated Tsp gene.

SUMMARY OF THE INVENTION

The present invention provides a recombinant gram-negative bacterialcell comprising a mutant spr gene encoding a mutant spr protein andwherein the cell comprises a non-recombinant wild-type chromosomal Tspgene.

In one embodiment, the genome of the cell according to the presentinvention is isogenic to the genome of a wild-type bacterial cell exceptfor the mutated spr gene.

The cells provided by the present invention show advantageous growth andprotein production phenotypes.

The present invention also provides a method for producing a recombinantprotein of interest comprising expressing the recombinant protein ofinterest in a recombinant gram-negative bacterial cell as defined above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the growth of MXE012 and MXE017 compared to the wild-typeW3110 and MXE001.

FIG. 2 shows the expression of the anti-TNFα Fab′ in MXE012 and MXE017compared to the wild-type W3110 and MXE001.

FIG. 3 shows the growth profile of W3110 and MXE012 during a anti-TNFαFab′ producing fermentation.

FIG. 4 shows periplasmic anti-TNFα Fab′ accumulation (filled lines andsymbols) and media Fab′ accumulation (dashed lines and open symbols) forW3110 and MXE012 (W3110 spr H119A) during a anti-TNFα Fab′ producingfermentation.

FIG. 5 shows the growth profile of anti-TNFα Fab′ expressing strainsW3110 and MXE012 and of anti-TNFα Fab′ and recombinant DsbC expressingstrains W3110 and MXE012.

FIG. 6 shows anti-TNFα Fab′ yield from the periplasm (shaded symbols)and supernatant (open unshaded symbols) from anti-TNFα Fab′ expressingstrains W3110 and MXE012 and of anti-TNFα Fab′ and recombinant DsbCexpressing strains W3110 and MXE012.

FIG. 7 shows the results of a dsDNA assay of strains W3110, MXE001,MXE008 and MXE012.

FIG. 8 shows the results of a protein assay of strains W3110, MXE001,MXE008 and MXE012.

FIG. 9 a shows the 5′ end of the wild type ptr (protease III) andknockout mutated ptr (protease III) protein and gene sequences.

FIG. 9 b shows the 5′ end of the wild type Tsp and knockout mutated Tspprotein and gene sequences.

FIG. 9 c shows a region of the wild type DegP and mutated DegP proteinand gene sequences.

FIG. 10 shows the construction of a vector for use in producing a cellaccording to an embodiment of the present invention.

FIG. 11 shows the growth profiles of 200 L fermentations of anti-TNFαFab′ and recombinant DsbC expressing strain MXE012.

FIG. 12 shows the anti-TNFα Fab′ titres of 200 L fermentations ofanti-TNFα Fab′ and recombinant DsbC expressing strain MXE012.

FIG. 13 shows the viabilities of 200 L fermentations of anti-TNFα Fab′and recombinant DsbC expressing strain MXE012.

FIG. 14 shows the growth profiles of 3000 L fermentations of anti-TNFαFab′ and recombinant DsbC expressing strain MXE012.

FIG. 15 shows the anti-TNFα Fab′ titres of 3000 L fermentations ofanti-TNFα Fab′ and recombinant DsbC expressing strain MXE012.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the DNA sequence of the wild-type Tsp gene including the6 nucleotides ATGAAC upstream of the start codon.

SEQ ID NO:2 is the amino acid sequence of the wild-type Tsp protein.

SEQ ID NO:3 is the DNA sequence of a mutated knockout Tsp gene includingthe 6 nucleotides ATGAAT upstream of the start codon.

SEQ ID NO:4 is the DNA sequence of the wild-type Protease III gene.

SEQ ID NO:5 is the amino acid sequence of the wild-type Protease IIIprotein.

SEQ ID NO:6 is the DNA sequence of a mutated knockout Protease III gene.

SEQ ID NO:7 is the DNA sequence of the wild-type DegP gene. SEQ ID NO:8is the amino acid sequence of the wild-type DegP protein.

SEQ ID NO:9 is the DNA sequence of a mutated DegP gene.

SEQ ID NO:10 is the amino acid sequence of a mutated. DegP protein.

SEQ ID NO: 11 is the amino acid sequence of the light chain variableregion of an anti-TNF antibody.

SEQ ID NO:12 is the amino acid sequence of the heavy chain variableregion of an anti-TNF antibody.

SEQ ID NO:13 is the amino acid sequence of the light chain of ananti-TNF antibody.

SEQ ID NO:14 is the amino acid sequence of the heavy chain of ananti-TNF antibody.

SEQ ID NO: 15 is the sequence of the 3′ oligonucleotide primer for theregion of the mutated Tsp gene comprising the Ase I restriction site.

SEQ ID NO: 16 is the sequence of the 5′ oligonucleotide primer for theregion of the mutated Tsp gene comprising the Ase I restriction site.

SEQ ID NO: 17 is the sequence of the 3′ oligonucleotide primer for theregion of the mutated Protease III gene comprising the Ase I restrictionsite.

SEQ ID NO: 18 is the sequence of the 5′ oligonucleotide primer for theregion of the mutated Protease III gene comprising the Ase I restrictionsite.

SEQ ID NO: 19 is the sequence of the 5′ oligonucleotide primer for theregion of the mutated DegP gene comprising the Ase I restriction site.

SEQ ID NO: 20 is the sequence of the 3′ oligonucleotide primer for theregion of the mutated DegP gene comprising the Ase I restriction site.

SEQ ID NO: 21 is the sequence of the wild-type spr gene including thesignal sequence which is the first 26 amino acid residues. SEQ ID NO:22is the sequence of the non-mutated spr gene without the signal sequence.

SEQ ID NO: 23 is the nucleotide sequence of a mutated OmpT sequencecomprising D210A and H212A mutations.

SEQ ID NO: 24 is the amino acid sequence of a mutated OmpT sequencecomprising D210A and H212A mutations.

SEQ ID NO: 25 is the nucleotide sequence of a mutated knockout OmpTsequence.

SEQ ID NO: 26 is the nucleotide sequence of his-tagged DsbC.

SEQ ID NO: 27 is the amino acid sequence of his-tagged DsbC.

SEQ ID NO: 28 shows the amino acid sequence of CDRH1 of hTNF40.

SEQ ID NO: 29 shows the amino acid sequence of CDRH2 of hTNF40 which isa hybrid CDR wherein the C-terminal six amino acids are from the H₂CDRsequence of a human subgroup 3 germline antibody and the amino acidchanges to the sequence resulting from this hybridisation are underlinedas follows: WINTYIGEPI YADSVKG.

SEQ ID NO: 30 shows the amino acid sequence of CDRH3 of hTNF40.

SEQ ID NO: 31 shows the amino acid sequence of CDRL1 of hTNF40.

SEQ ID NO: 32 shows the amino acid sequence of CDRL2 of hTNF40.

SEQ ID NO: 33 shows the amino acid sequence of CDRL3 of hTNF40.

SEQ ID NO: 34 shows the amino acid sequence of CDRH2 of hTNF40.

SEQ ID NO: 35 shows the sequence of the OmpA oligonucleotide adapter.

SEQ ID NO: 36 shows the oligonucleotide cassette encoding intergenicsequence 1 (IGS1) for E. coli Fab expression.

SEQ ID NO: 37 shows the oligonucleotide cassette encoding intergenicsequence 2 (IGS2) for E. coli Fab expression.

SEQ ID NO: 38 shows the oligonucleotide cassette encoding intergenicsequence 3 (IGS3) for E. coli Fab expression.

SEQ ID NO: 39 shows the oligonucleotide cassette encoding intergenicsequence 4 (IGS4) for E. coli Fab expression.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides a recombinant gram-negative bacterialcell suitable for expressing a protein of interest which comprises amutated spr gene and a non-recombinant wild-type chromosomal Tsp gene.

It has been surprisingly found that cells carrying a mutated spr and anon-recombinant wild-type chromosomal Tsp exhibit improved cell growthand exhibit reduced cell lysis phenotype compared to a wild-type cell ora cell comprising a mutated Tsp gene.

Further, in one embodiment cells carrying a mutant spr and anon-recombinant wild-type chromosomal Tsp exhibit increased yield of arecombinant protein of interest compared to a wild-type bacterial cellor a cell comprising a mutated Tsp gene. The improved protein yield maybe the periplasmic protein yield and/or the supernatant protein yield.In one embodiment the cells of the present invention show improvedperiplasmic protein yield compared to a wild-type cell due to reducedleakage from the cell. The recombinant bacterial cells are be capable ofprolonged expression of a recombinant protein of interest due to reducedcell lysis.

The cells according to the present invention preferably express amaximum yield in the periplasm and/or media of approximately 1.0 g/L,1.5 g/L, 1.8 g/L, 2.0 g/L, 2.4 g/L, 2.5 g/L, 3.0 g/L, 3.5 g/L or 4.0 g/Lof a protein of interest.

A drawback associated with known genetically engineered strains, such asthe protease deficient bacterial strains, previously created and used toexpress recombinant proteins involves the use of mutations of genesinvolved in cell metabolism and DNA replication such as, for examplephoA, fhuA, lac, rec, gal, ara, arg, thi and pro in E. coli strains.These mutations may have many deleterious effects on the host cellincluding effects on cell growth, stability, recombinant proteinexpression yield and toxicity. Strains having one or more of thesegenomic mutations, particularly strains having a high number of thesemutations, may exhibit a loss of fitness which reduces bacterial growthrate to a level which is not suitable for industrial protein production.Further, any of the above genomic mutations may affect other genes incis and/or in trans in unpredictable harmful ways thereby altering thestrain's phenotype, fitness and protein profile. Further, the use ofheavily mutated cells is not generally suitable for producingrecombinant proteins for commercial use, particularly therapeutics,because such strains generally have defective metabolic pathways andhence may grow poorly or not at all in minimal or chemically definedmedia.

In a preferred embodiment of the invention, the cells carry only theminimal mutations to the genome required to introduce the spr mutant. Inthis embodiment, the genome of the bacterial cell only differs from thegenome of a wild-type bacterial cell by one or more mutations to the sprgene and do not carry any other mutations which may have deleteriouseffects on the cell's growth and/or ability to express a protein ofinterest. Accordingly, one or more of the recombinant host cellsaccording to the present invention may exhibit improved proteinexpression and/or improved growth characteristics compared to cellscomprising further genetically engineered mutations to the genomicsequence. The cells provided by the present invention are also moresuitable for use to produce therapeutic proteins compared to cellscomprising further disruptions to the cell genome.

The present invention also provides a recombinant gram-negativebacterial cell comprising a mutant spr gene encoding a mutant sprprotein, wherein the genome of the cell is isogenic to the genome of awild-type bacterial cell except for the mutated spr gene. In this aspectof the present invention, the cell carries a wild-type Tsp gene. Thewild-type chromosomal Tsp gene is preferably a non-recombinantchromosomal Tsp gene.

The skilled person would easily be able to test a candidate cell cloneto see if it has the desired yield of a protein of interest usingmethods well known in the art including a fermentation method, ELISA andprotein G hplc. Suitable fermentation methods are described in HumphreysD P, et al. (1997). Formation of dimeric Fabs in E. coli: effect ofhinge size and isotype, presence of interchain disulphide bond, Fab′expression levels, tail piece sequences and growth conditions. J.IMMUNOL. METH. 209: 193-202; Backlund E. Reeks D. Markland K. Weir N.Bowering L. Larsson G. Fedbatch design for periplasmic product retentionin Escherichia coli, Journal Article. Research Support, Non-U.S. Gov'tJournal of Biotechnology. 135(4):358-65, 2008 Jul. 31; Champion KM.Nishihara JC. Joly JC. Arnott D. Similarity of the Escherichia coliproteome upon completion of different biopharmaceutical fermentationprocesses. [Journal Article] Proteomics. 1(9):1133-48, 2001 September;and Horn U. Strittmatter W. Krebber A. Knupfer U. Kujau M. Wenderoth R.Muller K. Matzku S. Pluckthun A. Riesenberg D. High volumetric yields offunctional dimeric miniantibodies in Escherichia coli, using anoptimized expression vector and high-cell-density fermentation undernon-limited growth conditions, Journal Article. Research Support,Non-U.S. Gov't Applied Microbiology & Biotechnology. 46(5-6):524-32,1996 Dec. The skilled person would also easily be able to test secretedprotein to see if the protein is correctly folded using methods wellknown in the art, such as protein G HPLC, circular dichroism, NMR, X-Raycrystallography and epitope affinity measurement methods.

In a preferred embodiment of the present invention, the cell furthercomprises a recombinant polynucleotide encoding DsbC.

The present invention will now be described in more detail.

The terms “protein” and “polypeptide” are used interchangeably herein,unless the context indicates otherwise. “Peptide” is intended to referto 10 or less amino acids.

The terms “polynucleotide” includes a gene, DNA, cDNA, RNA, mRNA etcunless the context indicates otherwise.

As used herein, the term “comprising” in context of the presentspecification should be interpreted as “including”.

The non-mutated cell or control cell in the context of the presentinvention means a cell of the same type as the recombinant gram-negativecell of the invention wherein the cell has not been modified to carrythe mutant spr gene. For example, a non-mutated cell may be a wild-typecell and may be derived from the same population of host cells as thecells of the invention before modification to introduce the anymutations.

The expressions “cell”, “cell line”, “cell culture” and “strain” areused interchangeably.

The expression “phenotype of a cell comprising a mutated Tsp gene” inthe context of the present invention means the phenotype exhibited by acell harbouring a mutant Tsp gene. Typically cells comprising a mutantTsp gene may lyse, especially at high cell densities. The lysis of thesecells causes any recombinant protein to leak into the supernatant. Cellscarrying mutated Tsp gene may also show thermosensitive growth at lowosmolarity. For example, the cells exhibit no or reduced growth rate orthe cells die in hypotonic media at a high temperature, such as at 40°C. or more.

The term “isogenic” in the context of the present invention means thatthe genome of the cell of the present invention has substantially thesame or the same genomic sequence compared to the wild-type cell fromwhich the cell is derived except for mutated spr gene. In thisembodiment the genome of the cell according to the present inventioncomprises no further non-naturally occurring or genetically engineeredmutations. In one embodiment the cell according to the present inventionmay have substantially the same genomic sequence compared to thewild-type cell except for the mutated spr gene, taking into account anynaturally occurring mutations which may occur. In one embodiment, thecell according to the present invention may have exactly the samegenomic sequence compared to the wild-type cell except for the mutatedspr gene.

In the embodiment of the present invention wherein the cell comprises arecombinant polynucleotide encoding DsbC, the polynucleotide encodingDsbC may be present on a suitable expression vector transformed into thecell and/or integrated into the host cell's genome. In the embodimentwhere the polynucleotide encoding DsbC is inserted into the host'sgenome, the cell of the present invention will also differ from awild-type cell due to the inserted polynucleotide sequence encoding theDsbC. Preferably the polynucleotide encoding DsbC is in an expressionvector in the cell thereby causing minimal disruption to the host cell'sgenome.

The term “wild-type” in the context of the present invention means astrain of a gram-negative bacterial cell as it may occur in nature ormay be isolated from the environment, which does not carry anygenetically engineered mutations. An example of a wild-type strain of E.coli is W3110, such as W3110 K-12 strain.

Any suitable gram-negative bacterium may be used as the parental cellfor producing the recombinant cell of the present invention. Suitablegram-negative bacterium include Salmonella typhimurium, Pseudomonasfluorescens, Erwinia carotovora, Shigella, Klebsiella pneumoniae,Legionella pneumophila, Pseudomonas aeruginosa, Acinetobacter baumanniiand E. coli. Preferably the parental cell is E. coli. Any suitablestrain of E. coli may be used in the present invention but preferably awild-type W3110 strain, such as K-12 W3110, is used.

In a preferred embodiment, the cell is isogenic to a wild-type E. colicell, such as W3110, except for the mutated spr gene.

In one embodiment the cell of the present invention comprises apolynucleotide encoding the protein of interest. In this embodiment, thepolynucleotide encoding the protein of interest may be contained withina suitable expression vector transformed into the cell and/or integratedinto the host cell's genome. In the embodiment where the polynucleotideencoding the protein of interest is inserted into the host's genome, thegenome of the present invention will also differ from a wild-type celldue to the inserted polynucleotide sequence encoding the protein ofinterest. Preferably the polynucleotide is in an expression vector inthe cell thereby causing minimal disruption to the host cell's genome.

The cells according to the present invention carry a wild-type Tsp gene.In one aspect of the present invention the cells carry a wild-typenon-recombinant chromosomal Tsp gene. The wild-type non-recombinantchromosomal Tsp gene refers to a chromosomal Tsp gene that is notconstructed, produced or inserted into the chromosome using recombinantDNA technology.

As used herein, “Tsp gene” means a gene encoding protease Tsp (alsoknown as Pre) which is a periplasmic protease capable of acting onPenicillin-binding protein-3 (PBP3) and phage tail proteins. Thesequence of the wild-type Tsp gene is shown in SEQ ID NO: 1 and thesequence of the wild-type Tsp protein is shown in SEQ ID NO: 2.

The spr protein is a 18 kDa membrane bound periplasmic protease and thesubstrates of spr are Tsp and peptidoglycans in the outer membraneinvolved in cell wall hydrolysis during cell division.

The wild-type amino acid sequence of the spr protein is shown in SEQ IDNO:21 with the signal sequence at the N-terminus and in SEQ ID NO:22without the signal sequence of 26 amino acids (according to UniProtAccession Number POAFV4). The amino acid numbering of the spr proteinsequence in the present invention includes the signal sequence.Accordingly, the amino acid 1 of the spr protein is the first amino acid(Met) shown in SEQ ID NO: 21.

The mutated spr gene is preferably the cell's chromosomal spr gene.

The mutated spr gene encodes a spr protein capable of suppressing thephenotype of a cell comprising a mutated Tsp gene. Cells carryingmutated Tsp gene may have a good cell growth rate but one limitation ofthese cells is their tendency to lyse, especially at high celldensities. Accordingly the phenotype of a cell comprising a mutated Tspgene is a tendency to lyse, especially at high cell densities. Cellscarrying mutated Tsp gene also show thermosensitive growth at lowosmolarity. However, the spr mutations carried by the cells of thepresent invention, when introduced into a cell carrying a mutated Tspgene suppress the mutant Tsp phenotype and, therefore, the cell exhibitsreduced lysis, particularly at a high cell density. The growth phenotypeof a cell may be easily measured by a person skilled in the art duringshake flask or high cell density fermentation technique. The suppressionof the cell lysis phenotype may be been seen from the improved growthrate and/or recombinant protein production, particularly in theperiplasm, exhibited by a cell carrying spr mutant and Tsp mutantcompared to a cell carrying the Tsp mutant and a wild-type spr.

Any suitable mutation or mutations may be made to the spr gene whichresults in a spr protein capable of suppressing the phenotype of a cellcomprising a mutated Tsp gene. This activity may be tested by a personskilled in the art by creating a cell carrying a mutant spr gene andmutant Tsp gene and comparing the phenotype to a cell carrying themutant Tsp gene only. Suitable mutations to the Tsp gene are describedin detail below. Reference to a mutated Tsp gene or mutated Tsp geneencoding Tsp, refers to either a mutated Tsp gene encoding a Tsp proteinhaving reduced protease activity or a knockout mutated Tsp gene, unlessotherwise indicated.

The expression “mutated Tsp gene encoding a Tsp protein having reducedprotease activity” means that the mutated Tsp gene does not have thefull protease activity compared to the wild-type non-mutated Tsp gene.The mutated Tsp gene may encode a Tsp protein having 50% or less, 40% orless, 30% or less, 20% or less, 10% or less or 5% or less of theprotease activity of a wild-type non-mutated Tsp protein. The mutatedTsp gene may encode a Tsp protein having no protease activity. The cellis not deficient in chromosomal Tsp i.e. the Tsp gene sequence has notbeen deleted or mutated to prevent expression of any form of Tspprotein.

Any suitable mutation may be introduced into the Tsp gene in order toproduce a protein having reduced protease activity. The proteaseactivity of a Tsp protein expressed from a gram-negative bacterium maybe easily tested by a person skilled in the art by any suitable methodin the art, such as the method described in Keiler et al (Identificationof Active Site Residues of the Tsp Protease* THE JOURNAL OF BIOLOGICALCHEMISTRY Vol. 270, No. 48, Issue of December 1, pp. 28864-28868, 1995Kenneth C. Keiler and Robert T. Sauer) wherein the protease activity ofTsp was tested.

Tsp has been reported in Keiler et al (supra) as having an active sitecomprising residues S430, D441 and K455 and residues G375, G376, E433and T452 are important for maintaining the structure of Tsp. Keiler etal (supra) reports findings that the mutated Tsp genes S430A, D441A,K455A, K455H, K455R, G375A, G376A, E433A and T452A had no detectableprotease activity. It is further reported that the mutated Tsp geneS430C displayed about 5-10% wild-type activity. Accordingly, the Tspmutation to produce a protein having reduced protease activity maycomprise a mutation, such as a missense mutation to one or more ofresidues S430, D441, K455, G375, G376, E433 and T452. Preferably the Tspmutation to produce a protein having reduced protease activity maycomprise a mutation, such as a missense mutation to one, two or allthree of the active site residues 5430, D441 and K455.

Accordingly the mutated Tsp gene may comprise:

-   -   a mutation to 5430; or    -   a mutation to D441; or    -   a mutation to K455; or    -   a mutation to S430 and D441; or    -   a mutation to 5430 and K455; or    -   a mutation to D441 and K455; or    -   a mutation to S430, D441 and K455.

One or more of S430, D441, K455, G375, G376, E433 and T452 may bemutated to any suitable amino acid which results in a protein havingreduced protease activity. Examples of suitable mutations are S430A,S430C, D441A, K455A, K455H, K455R, G375A, G376A, E433A and T452A. Themutated Tsp gene may comprise one, two or three mutations to the activesite residues, for example the gene may comprise:

-   -   S430A or S430C; and/or    -   D441A; and/or    -   K455A or K45514 or K455R.

Preferably, the Tsp gene comprises the point mutation S430A or S430C.

The expression “knockout mutated Tsp gene” means that the gene comprisesone or more mutations thereby causing no expression of the proteinencoded by the gene to provide a cell deficient in the protein encodedby the knockout mutated gene. The knockout gene may be partially orcompletely transcribed but not translated into the encoded protein. Theknockout mutated Tsp gene may be mutated in any suitable way, forexample by one or more deletion, insertion, point, missense, nonsenseand frameshift mutations, to cause no expression of the protein. Forexample, the gene may be knocked out by insertion of a foreign DNAsequence, such as an antibiotic resistance marker, into the gene codingsequence.

The mutated Tsp gene may comprises a mutation to the gene start codonand/or one or more stop codons positioned downstream of the gene startcodon and upstream of the gene stop codon thereby preventing expressionof the Tsp protein. The mutation to the start codon may be a missensemutation of one, two or all three of the nucleotides of the start codon.Alternatively or additionally the start codon may be mutated by aninsertion or deletion frameshift mutation. The Tsp gene comprises twoATG codons at the 5′ end of the coding sequence, one or both of the ATGcodons may be mutated by a missense mutation. The Tsp gene may bemutated at the second ATG codon (codon 3) to TCG, as shown in FIG. 9 b.The Tsp gene may alternatively or additionally comprise one or more stopcodons positioned downstream of the gene start codon and upstream of thegene stop codon. Preferably the knockout mutated Tsp gene comprises botha missense mutation to the start codon and one or more inserted stopcodons. The Tsp gene may be mutated to delete “T” from the fifth codonthereby causing a frameshift resulting in stop codons at codons 11 and16, as shown in FIG. 9 b. The Tsp gene may be mutated to insert an Ase Irestriction site to create a third in-frame stop codon at codon 21, asshown in FIG. 9 b.

The knockout mutated Tsp gene may have the DNA sequence of SEQ ID NO: 3,which includes the 6 nucleotides ATGAAT upstream of the start codon. Themutations which have been made in the knockout mutated Tsp sequence ofSEQ ID NO: 3 are shown in FIG. 9 b. In one embodiment the mutated Tspgene has the DNA sequence of nucleotides 7 to 2048 of SEQ ID NO:3.

Accordingly, once a cell carrying a suitable mutant Tsp gene has beenidentified, suitable spr gene mutations can be identified which resultsin a spr protein capable of suppressing the phenotype of a cellcomprising a mutated Tsp gene.

The cells according to a preferred embodiment of the present inventioncomprise a mutant spr gene encoding a spr protein having a mutation atone or more amino acids selected from N31, R62, I70, Q73, C94, S95, V98,Q99, R100, L108, Y115, D133, V135, L136, G140, R144, H145, G147, H157and W174, more preferably at one or more amino acids selected from C94,S95, V98, Y115, D133, V135, H145, G147, H157 and W174. In thisembodiment, the spr protein preferably does not have any furthermutations. Preferably the mutant spr gene encodes a spr protein having amutation at one or more amino acids selected from N31, R62, I70, Q73,C94, S95, V98, Q99, R100, L108, Y115, D133, V135, L136, G140, R144,H145, G147 and H157, more preferably at one or more amino acids selectedfrom C94, S95, V98, Y115, D133, V135, H145, G147 and H157. In thisembodiment, the spr protein preferably does not have any furthermutations. Preferably, the mutant spr gene encodes a spr protein havinga mutation at one or more amino acids selected from N31, R62, I70, Q73,S95, V98, Q99, R100, L108, Y115, D133, V135, L136, G140, R144 and G147,more preferably at one or more amino acids selected from S95, V98, Y115,D133, V135 and G147. In this embodiment, the spr protein preferably doesnot have any further mutations.

In one aspect of the present invention there is provided a gram-negativebacterial cell comprising a mutant spr gene encoding a spr proteinhaving a mutation at one or more amino acids selected from C94, S95,V98, Y115, D133, V135, H145, G147 and H157, preferably at one or moreamino acids selected from S95, V98, Y115, D133, V135 and G147, andwherein the cell comprises a wild-type Tsp gene. In this embodiment, thespr protein preferably does not have any further mutations.

The wild-type chromosomal Tsp gene is preferably a non-recombinantchromosomal Tsp gene. Preferably, the cell further comprises arecombinant polynucleotide encoding DsbC.

The mutation to one or more of the above amino acids may be any suitablemissense mutation to one, two or three of the nucleotides encoding theamino acid. The mutation changes the amino acid residue to any suitableamino acid which results in a mutated spr protein capable of suppressingthe phenotype of a cell comprising a mutated Tsp gene. The missensemutation may change the amino acid to one which is a different sizeand/or has different chemical properties compared to the wild-type aminoacid.

In one embodiment the mutation is to one, two or three of the catalytictriad of amino acid residues of C94, H145, and H157 (Solution NMRStructure of the N1 μC/P60 Domain of Lipoprotein Spr from Escherichiacoli Structural Evidence for a Novel Cysteine Peptidase Catalytic Triad,Biochemistry, 2008, 47, 9715-9717).

Accordingly, the mutated spr gene may comprise:

-   -   a mutation to C94; or    -   a mutation to H145; or    -   a mutation to H157; or    -   a mutation to C94 and H145; or    -   a mutation to C94 and H157; or    -   a mutation to H145 and H157; or    -   a mutation to C94, H145 and H157.

In this embodiment, the spr protein preferably does not have any furthermutations.

One, two or three of C94, H145 and H157 may be mutated to any suitableamino acid which results in a spr protein capable of suppressing thephenotype of a cell comprising a mutated Tsp gene. For example, one, twoor three of C94, H145, and H157 may be mutated to a small amino acidsuch as Gly or Ala. Accordingly, the spr protein may have one, two orthree of the mutations C94A, H145A and H157A. Preferably, the spr genecomprises the missense mutation H145A, which has been found to produce aspr protein capable of suppressing the phenotype of a cell comprising amutated Tsp gene.

The designation for a substitution mutant herein consists of a letterfollowed by a number followed by a letter. The first letter designatesthe amino acid in the wild-type protein. The number refers to the aminoacid position where the amino acid substitution is being made, and thesecond letter designates the amino acid that is used to replace thewild-type amino acid.

In a preferred embodiment the mutant spr protein comprises a mutation atone or more amino acids selected from N31, R62, I70, Q73, S95, V98, Q99,R100, L108, Y115, D133, V135, L136, G140, R144 and G147, preferably amutation at one or more amino acids selected from S95, V98, Y115, D133,V135 and G147. In this embodiment, the spr protein preferably does nothave any further mutations. Accordingly, the mutated spr gene maycomprise:

-   -   a mutation to N31; or    -   a mutation to R62; or    -   a mutation to 170; or    -   a mutation to Q73; or    -   a mutation to S95; or    -   a mutation to V98; or    -   a mutation to Q99; or    -   a mutation to R100; or    -   a mutation to L108; or    -   a mutation to Y115; or    -   a mutation to D133; or    -   a mutation to V135; or    -   a mutation to L136; or    -   a mutation to G140; or    -   a mutation to R144; or    -   a mutation to G147.

In one embodiment the mutant spr protein comprises multiple mutations toamino acids:

-   -   S95 and Y115; or    -   N31, Q73, R100 and G140; or    -   Q73, R100 and G140; or    -   R100 and G140; or    -   Q73 and G140; or    -   Q73 and R100; or    -   R62, Q99 and R144; or    -   Q99 and R144.

One or more of the amino acids N31, R62, I70, Q73, S95, V98, Q99, R100,L108, Y115, D133, V135, L136, G140, R144 and G147 may be mutated to anysuitable amino acid which results in a spr protein capable ofsuppressing the phenotype of a cell comprising a mutated Tsp gene. Forexample, one or more of N31, R62, I70, Q73, S95, V98, Q99, R100, L108,Y115, D133, V135, L136, G140 and R144 may be mutated to a small aminoacid such as Gly or Ala.

In a preferred embodiment the spr protein comprises one or more of thefollowing mutations: N31Y, R62c, 170T, Q73R, S95F, V98E, Q99P, R100G,L108S, Y115F, D133A, V135D or V135G, L136P, G140C, R144c and G147C.Preferably the spr protein comprises one or more of the followingmutations: S95F, V98E, Y115F, D133A, V135D or V135G and G147C. In thisembodiment, the spr protein preferably does not have any furthermutations.

In one embodiment the spr protein has one mutation selected from N31Y,R62c, 170T, Q73R, S95F, V98E, Q99P, R100G, L108S, Y115F, D133A, V135D orV135G, L136P, G140C, R144c and G147C. In this embodiment, the sprprotein preferably does not have any further mutations.

In a further embodiment the spr protein has multiple mutations selectedfrom:

-   -   S95F and Y115F    -   N31Y, Q73R, R100G and G140C;    -   Q73R, R100G and G140C;    -   R100G and G140C;    -   Q73R and G140C;    -   Q73R and R100G;    -   R62c, Q99P and R144c; or    -   Q99P and R144c.

In one embodiment the spr protein has the mutation W174R. In analternative embodiment the spr protein does not have the mutation W174R.

In a preferred embodiment the cell according to the present inventioncomprises the mutated spr gene and a recombinant polynucleotide encodingDsbC.

As used herein, a “recombinant polypeptide” refers to a protein that isconstructed or produced using recombinant DNA technology. Thepolynucleotide sequence encoding DsbC may be identical to the endogenoussequence encoding DsbC found in bacterial cells. Alternatively, therecombinant polynucleotide sequence encoding DsbC is a mutated versionof the wild-type DsbC sequence, for example having a restriction siteremoved, such as an EcoRI site, and/or a sequence encoding a his-tag. Anexample modified DsbC nucleotide sequence for use in the presentinvention is shown in SEQ ID NO: 26, which encodes the his-tagged DsbCamino acid sequence shown in SEQ ID NO: 27.

In one aspect of the present invention there is provided a gram-negativebacterial cell comprising a mutant spr gene encoding a mutant sprprotein, a recombinant polynucleotide encoding DsbC and wherein the cellcomprises a wild-type Tsp gene. The wild-type Tsp gene is preferably anon-recombinant chromosomal Tsp gene.

DsbC is a prokaryotic protein found in the periplasm of E. coli whichcatalyzes the formation of disulphide bonds in E. coli. DsbC has anamino acid sequence length of 236 (including signal peptide) and amolecular weight of 25.6 KDa (UniProt No. POAEG6). DsbC was firstidentified in 1994 (Missiakas et al. The Escherichia coli dsbC (xprA)gene encodes a periplasmic protein involved in disulfide bond formation,The EMBO Journal vol 13, no 8, p 2013-2020, 1994 and Shevchik et al.Characterization of DsbC, a periplasmic protein of Erwinia chrysanthemiand Escherichia coli with disulfide isomerase activity, The EMBO Journalvol 13, no 8, p 2007-2012, 1994).

It is known to co-express proteins which catalyze the formation ofdisulphide bonds to improve protein expression in a host cell.WO98/56930 discloses a method for producing heterologous disulfidebond-containing polypeptides in bacterial cells wherein a prokaryoticdisulfide isomerase, such as DsbC or DsbG is co-expressed with aeukaryotic polypeptide. U.S. Pat. No. 6,673,569 discloses an artificialoperon comprising polynucleotides encoding each of DsbA, DsbB, DsbC andDsbD for use in producing a foreign protein. EP0786009 discloses aprocess for producing a heterologous polypeptide in bacteria wherein theexpression of nucleic acid encoding DsbA or DsbC is induced prior to theinduction of expression of nucleic acid encoding the heterologouspolypeptide.

We have found that the specific combination of the expression ofrecombinant polynucleotide encoding DsbC in a bacterial cell whichcomprises a mutated spr gene and a wild-type Tsp gene provides animproved host for expressing proteins of interest. It was surprisinglyfound that the new strains exhibit increased cell growth rate andincreased cell survival duration compared to a wild-type cell or a cellcomprising a mutated Tsp gene. Specifically, cells carrying arecombinant DsbC gene, a spr mutation and a wild-type Tsp exhibitreduced cell lysis phenotype compared to cells carrying a mutated Tspgene.

In one embodiment the cell according to the present invention alsoexpresses one or more further proteins as follows:

-   -   one or more proteins capable of facilitating protein folding,        such as FkpA, Skp, SurA, PPiA and PPiD; and/or    -   one or more protein capable of facilitating protein secretion or        translocation, such as SecY, SecE, SecG, SecYEG, SecA, SecB,        FtsY and Lep; and/or    -   one or more proteins capable of facilitating disulphide bond        formation, such as DsbA, DsbB, DsbD, DsbG.

One of more of the above proteins may be integrated into the cell'sgenome and/or inserted in an expression vector.

In one embodiment the cell according to the present invention does notcomprise recombinant polynucleotide encoding one or more of thefollowing further proteins:

-   -   one or more proteins capable of facilitating protein folding,        such as FkpA, Skp, SurA, PPiA and PPiD;    -   one or more protein capable of facilitating protein secretion or        translocation, such as SecY, SecE, SecG, SecYEG, SecA, SecB,        FtsY and Lep; and    -   one or more proteins capable of facilitating disulphide bond        formation, such as DsbA, DsbB, DsbD, DsbG.

In a preferred embodiment of the present invention the recombinantgram-negative bacterial cell further comprises a mutated DegP geneencoding a DegP protein having chaperone activity and reduced proteaseactivity and/or a mutated ptr gene, wherein the mutated ptr gene encodesa Protease III protein having reduced protease activity or is a knockoutmutated ptr gene and/or a mutated OmpT gene, wherein the mutated OmpTgene encodes an OmpT protein having reduced protease activity or is aknockout mutated OmpT gene.

In one embodiment the present invention provides a recombinantgram-negative bacterial cell comprising

a. a mutated spr gene;

b. a wild-type non-recombinant chromosomal Tsp gene; and

c. a mutated DegP gene encoding a DegP protein having chaperone activityand reduced protease activity and/or a mutated OmpT wherein the mutatedOmpT gene encodes an OmpT protein having reduced protease activity or isa knockout mutated OmpT gene.

Preferably in this embodiment the cell is isogenic to a wild-typebacterial cell except for the above mutations.

In one embodiment the present invention provides a recombinantgram-negative bacterial cell comprising:

a. a mutated spr gene;

b. a wild-type non-recombinant chromosomal Tsp gene; and

c. a mutated ptr gene, wherein the mutated ptr gene encodes a ProteaseIII protein having reduced protease activity or is a knockout mutatedptr gene and/or a mutated OmpT wherein the mutated OmpT gene encodes anOmpT protein having reduced protease activity or is a knockout mutatedOmpT gene.

Preferably in this embodiment the cell is isogenic to a wild-typebacterial cell except for the above mutations.

In one embodiment the present invention provides a cell comprising

a. a mutated spr gene;

b. a wild-type non-recombinant chromosomal Tsp gene;

c. a mutated DegP gene encoding a DegP protein having chaperone activityand reduced protease activity;

d. a mutated ptr gene, wherein the mutated ptr gene encodes a ProteaseIII protein having reduced protease activity or is a knockout mutatedptr gene; and

e. optionally a mutated OmpT wherein the mutated OmpT gene encodes an

OmpT protein having reduced protease activity or is a knockout mutatedOmpT gene.

Preferably in this embodiment the cell is isogenic to a wild-typebacterial cell except for the above mutations.

In one embodiment of the present invention the cell carries a mutatedDegP gene. As used herein, “DegP” means a gene encoding DegP protein(also known as HtrA), which has dual function as a chaperone and aprotease (Families of serine peptidases; Rawlings N D, Barrett A J.Methods Enzymol. 1994; 244:19-61). The sequence of the non-mutated DegPgene is shown in SEQ ID NO: 7 and the sequence of the non-mutated DegPprotein is shown in SEQ ID NO: 8.

At low temperatures DegP functions as a chaperone and at hightemperatures DegP has a preference to function as a protease (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M) and The proteolytic activity of the HtrA(DegP) protein from Escherichia coli at low temperatures, Skorko-GlonekJ et al Microbiology 2008, 154, 3649-3658).

In the embodiments where the cell comprises the DegP mutation the DegPmutation in the cell provides a mutated DegP gene encoding a DegPprotein having chaperone activity but not full protease activity.

The expression “having chaperone activity” in the context of the presentinvention means that the mutated DegP protein has the same orsubstantially the same chaperone activity compared to the wild-typenon-mutated DegP protein. Preferably, the mutated DegP gene encodes aDegP protein having 50% or more, 60% or more, 70% or more, 80% or more,90% or more or 95% or more of the chaperone activity of a wild-typenon-mutated DegP protein. More preferably, the mutated DegP gene encodesa DegP protein having the same chaperone activity compared to wild-typeDegP.

The expression “having reduced protease activity” in the context of thepresent invention means that the mutated DegP protein does not have thefull protease activity compared to the wild-type non-mutated DegPprotein. Preferably, the mutated DegP gene encodes a DegP protein having50% or less, 40% or less, 30% or less, 20% or less, 10% or less or 5% orless of the protease activity of a wild-type non-mutated DegP protein.More preferably, the mutated DegP gene encodes a DegP protein having noprotease activity. The cell is not deficient in chromosomal DegP i.e.the DegP gene sequences has not been deleted or mutated to preventexpression of any form of DegP protein.

Any suitable mutation may be introduced into the DegP gene in order toproduce a protein having chaperone activity and reduced proteaseactivity. The protease and chaperone activity of a DegP proteinexpressed from a gram-negative bacterium may be easily tested by aperson skilled in the art by any suitable method such as the methoddescribed in Spiess et al wherein the protease and chaperone activitiesof DegP were tested on MalS, a natural substrate of DegP (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M) and also the method described in Theproteolytic activity of the HtrA (DegP) protein from Escherichia coli atlow temperatures, Skorko-Glonek J et al Microbiology 2008, 154,3649-3658.

DegP is a serine protease and has an active center consisting of acatalytic triad of amino acid residues of His105, Asp135 and Ser210(Families of serine peptidases, Methods Enzymol., 1994, 244:19-61Rawlings N and Barrett A). The DegP mutation to produce a protein havingchaperone activity and reduced protease activity may comprise amutation, such as a missense mutation to one, two or three of His105,Asp135 and Ser210.

Accordingly, the mutated DegP gene may comprise:

-   -   a mutation to His105; or    -   a mutation to Asp135; or    -   a mutation to Ser210; or    -   a mutation to His105 and Asp135; or    -   a mutation to His105 and Ser210; or    -   a mutation to Asp135 and Ser210; or    -   a mutation to His105, Asp135 and Ser210.

One, two or three of His105, Asp135 and Ser210 may be mutated to anysuitable amino acid which results in a protein having chaperone activityand reduced protease activity. For example, one, two or three of His105,Asp135 and Ser210 may be mutated to a small amino acid such as Gly orAla. A further suitable mutation is to change one, two or three ofHis105, Asp135 and Ser210 to an amino acid having opposite propertiessuch as Asp135 being mutated to Lys or Arg, polar His105 being mutatedto a non-polar amino acid such as Gly, Ala, Val or Leu and smallhydrophilic Ser210 being mutated to a large or hydrophobic residue suchas Val, Leu, Phe or Tyr. Preferably, the DegP gene comprises the pointmutation S210A, as shown in FIG. 9 c, which has been found to produce aprotein having chaperone activity but not protease activity (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M).

DegP has two PDZ domains, PDZ1 (residues 260-358) and PDZ2 (residues359-448), which mediate protein-protein interaction (ATemperature-Dependent Switch from Chaperone to Protease in a WidelyConserved Heat Shock Protein. Cell, Volume 97, Issue 3, Pages 339-347.Spiess C, Beil A, Ehrmann M). In one embodiment of the present inventionthe degP gene is mutated to delete PDZ1 domain and/or PDZ2 domain. Thedeletion of PDZ1 and PDZ2 results in complete loss of protease activityof the DegP protein and lowered chaperone activity compared to wild-typeDegP protein whilst deletion of either PDZ1 or PDZ2 results in 5%protease activity and similar chaperone activity compared to wild-typeDegP protein (A Temperature-Dependent Switch from Chaperone to Proteasein a Widely Conserved Heat Shock Protein. Cell, Volume 97, Issue 3,Pages 339-347. Spiess C, Beil A, Ehrmann M).

The mutated DegP gene may also comprise a silent non-naturally occurringrestriction site, such as Ase I in order to aid in identification andscreening methods, for example as shown in FIG. 9 c.

The preferred sequence of the mutated DegP gene comprising the pointmutation S210A and an Ase I restriction marker site is provided in SEQID NO: 9 and the encoded protein sequence is shown in SEQ ID NO: 10. Themutations which have been made in the mutated DegP sequence of SEQ IDNO: 9 are shown in FIG. 9 c.

In the embodiments of the present invention wherein the cell comprises amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity, one or more of the cells provided by thepresent invention may provide improved yield of correctly foldedproteins from the cell relative to mutated cells wherein the DegP genehas been mutated to knockout DegP preventing DegP expression, such aschromosomal deficient DegP. In a cell comprising a knockout mutated DegPgene preventing DegP expression, the chaperone activity of DegP is lostcompletely whereas in the cell according to the present invention thechaperone activity of DegP is retained whilst the full protease activityis lost. In these embodiments, one or more cells according to thepresent invention have a lower protease activity to prevent proteolysisof the protein whilst maintaining the chaperone activity to allowcorrect folding and transportation of the protein in the host cell.

The skilled person would easily be able to test secreted protein to seeif the protein is correctly folded using methods well known in the art,such as protein G HPLC, circular dichroism, NMR, X-Ray crystallographyand epitope affinity measurement methods.

In these embodiments, one or more cells according to the presentinvention may have improved cell growth compared to cells carrying amutated knockout DegP gene preventing DegP expression. Without wishingto be bound by theory improved cell growth maybe exhibited due to theDegP protease retaining chaperone activity which may increase capacityof the cell to process all proteins which require chaperone activity.Accordingly, the production of correctly folded proteins necessary forthe cell's growth and reproduction may be increased in one or more ofthe cells of the present invention compared to cells carrying a DegPknockout mutation thereby improving the cellular pathways regulatinggrowth. Further, known DegP protease deficient strains are generallytemperature-sensitive and do not typically grow at temperatures higherthan about 28° C. However, the cells according to the present inventionare not temperature-sensitive and may be grown at temperatures of 28° C.or higher, including temperatures of approximately 30° C. toapproximately 37° C., which are typically used for industrial scaleproduction of proteins from bacteria.

In one embodiment of the present invention the cell carries a mutatedptr gene. As used herein, “ptr gene” means a gene encoding Protease III,a protease which degrades high molecular weight proteins. The sequenceof the non-mutated ptr gene is shown in SEQ ID NO: 4 and the sequence ofthe non-mutated Protease III protein is shown in SEQ ID NO: 5.

Reference to the mutated ptr gene or mutated ptr gene encoding ProteaseIII, refers to either a mutated ptr gene encoding a Protease III proteinhaving reduced protease activity or a knockout mutated ptr gene, unlessotherwise indicated.

The expression “mutated ptr gene encoding a Protease III protein havingreduced protease activity” in the context of the present invention meansthat the mutated ptr gene does not have the full protease activitycompared to the wild-type non-mutated ptr gene.

Preferably, the mutated ptr gene encodes a Protease III having 50% orless, 40% or less, 30% or less, 20% or less, 10% or less or 5% or lessof the protease activity of a wild-type non-mutated Protease IIIprotein. More preferably, the mutated ptr gene encodes a Protease IIIprotein having no protease activity. In this embodiment the cell is notdeficient in chromosomal ptr i.e. the ptr gene sequence has not beendeleted or mutated to prevent expression of any form of Protease IIIprotein.

Any suitable mutation may be introduced into the ptr gene in order toproduce a Protease III protein having reduced protease activity. Theprotease activity of a Protease III protein expressed from agram-negative bacterium may be easily tested by a person skilled in theart by any suitable method in the art.

The expression “knockout mutated ptr gene” in the context of the presentinvention means that the gene comprises one or more mutations therebycausing no expression of the protein encoded by the gene to provide acell deficient in the protein encoded by the knockout mutated gene. Theknockout gene may be partially or completely transcribed but nottranslated into the encoded protein. The knockout mutated ptr gene maybe mutated in any suitable way, for example by one or more deletion,insertion, point, missense, nonsense and frameshift mutations, to causeno expression of the protein. For example, the gene may be knocked outby insertion of a foreign DNA sequence, such as an antibiotic resistancemarker, into the gene coding sequence.

In a preferred embodiment the gene is not mutated by insertion of aforeign DNA sequence, such as an antibiotic resistance marker, into thegene coding sequence. Preferably the Protease III gene comprise amutation to the gene start codon and/or one or more stop codonspositioned downstream of the gene start codon and upstream of the genestop codon thereby preventing expression of the Protease III protein.

A mutation to the target knockout gene start codon causes loss offunction of the start codon and thereby ensures that the target genedoes not comprise a suitable start codon at the start of the codingsequence. The mutation to the start codon may be a missense mutation ofone, two or all three of the nucleotides of the start codon.Alternatively or additionally the start codon may be mutated by aninsertion or deletion frameshift mutation.

In a preferred embodiment the ptr gene is mutated to change the ATGstart codon to ATT, as shown in FIG. 9 a.

The knockout mutated ptr gene may alternatively or additionally compriseone or more stop codons positioned downstream of the gene start codonand upstream of the gene stop codon. Preferably the knockout mutated ptrgene comprises both a missense mutation to the start codon and one ormore inserted stop codons.

The one or more inserted stop codons are preferably in-frame stopcodons. However the one or more inserted stop codons may alternativelyor additionally be out-of-frame stop codons. One or more out-of-framestop codons may be required to stop translation where an out-of-framestart codon is changed to an in-frame start codon by an insertion ordeletion frameshift mutation. The one or more stop codons may beintroduced by any suitable mutation including a nonsense point mutationand a frameshift mutation. The one or more stop codons are preferablyintroduced by a frameshift mutation and/or an insertion mutation,preferably by replacement of a segment of the gene sequence with asequence comprising a stop codon. For example an Ase I restriction sitemay be inserted, which comprises the stop codon TAA.

In a preferred embodiment the ptr gene is mutated to insert an in-framestop codon by insertion of an Ase I restriction site, as shown in FIG. 9a. In a preferred embodiment the knockout mutated ptr gene has the DNAsequence of SEQ ID NO: 6. The mutations which have been made in theknockout mutated ptr gene sequence of SEQ ID NO: 6 are shown in FIG. 9a.

The above described knockout mutations are advantageous because theycause minimal or no disruption to the chromosomal DNA upstream ordownstream of the target knockout gene site and do not require theinsertion and retention of foreign DNA, such as antibiotic resistancemarkers, which may affect the cell's suitability for expressing aprotein of interest, particularly therapeutic proteins. Accordingly, oneor more of the cells according to the present invention may exhibitimproved growth characteristics and/or protein expression compared tocells wherein the protease gene has been knocked out by insertion offoreign DNA into the gene coding sequence.

In one embodiment the cells according to the present invention carry amutated OmpT gene. As used herein, “OmpT gene” means a gene encodingprotease OmpT (outer membrane protease T) which is an outer membraneprotease. The sequence of the wild-type non-mutated OmpT gene isSWISS-PROT P09169.

Reference to a mutated OmpT gene or mutated OmpT gene encoding OmpT,refers to either a mutated OmpT gene encoding a OmpT protein havingreduced protease activity or a knockout mutated OmpT gene, unlessotherwise indicated.

The expression “mutated OmpT gene encoding a OmpT protein having reducedprotease activity” in the context of the present invention means thatthe mutated OmpT gene does not have the full protease activity comparedto the wild-type non-mutated OmpT gene. The mutated OmpT gene may encodea OmpT protein having 50% or less, 40% or less, 30% or less, 20% orless, 10% or less or 5% or less of the protease activity of a wild-typenon-mutated OmpT protein. The mutated OmpT gene may encode a OmpTprotein having no protease activity. In this embodiment the cell is notdeficient in chromosomal OmpT i.e. the OmpT gene sequence has not beendeleted or mutated to prevent expression of any form of OmpT protein.

Any suitable mutation may be introduced into the OmpT gene in order toproduce a protein having reduced protease activity. The proteaseactivity of a OmpT protein expressed from a gram-negative bacterium maybe easily tested by a person skilled in the art by any suitable methodin the art, such as the method described in Kramer et al (Identificationof essential acidic residues of outer membrane protease OmpT supports anovel active site, FEBS Letters 505 (2001) 426-430) and Dekker et al(Substrate Specitificity of the Integral Membrane Protease OmpTDetermined by Spatially Addressed Peptide Libraries, Biochemistry 2001,40, 1694-1701).

OmpT has been reported in Kramer et al (Identification of active siteserine and histidine residues in Escherichia coli outer membraneprotease OmpT FEBS Letters 2000 468, 220-224) discloses thatsubstitution of serines, histidines and acidic residues by alaninesresults in ˜10-fold reduced activity for G1u27, Asp97, Asp208 or His101,˜500-fold reduced activity for Ser99 and ˜10000-fold reduced activityfor Asp83, Asp85, Asp210 or His212. Vandeputte-Rutten et al (CrystalStructure of the Outer Membrane Protease OmpT from Escherichia colisuggests a novel catalytic site, The EMBO Journal 2001, Vol 20 No. 185033-5039) as having an active site comprising a Asp83-Asp85 pair and aHis212-Asp210 pair. Further Kramer et al (Lipopolysaccharide regionsinvolved in the activation of Escherichia coli outer membrane proteaseOmpT, Eur. J. Biochem. FEBS 2002, 269, 1746-1752) discloses thatmutations D208A, D210A, H212A, H212N, H212Q, G216K/K217G, K217T andR218L in loop L4 all resulted in partial or virtually complete loss ofenzymatic activity.

Accordingly, the OmpT mutation to produce a protein having reducedprotease activity may comprise a mutation, such as a missense mutationto one or more of residues E27, D43, D83, D85, D97, S99, H101 E111,E136, E193, D206, D208, D210, H212 G216, K217, R218 & E250.

One or more of E27, D43, D83, D85, D97, S99, H101 E111, E136, E193,D206, D208, D210, H212 G216, K217, R218 & E250 may be mutated to anysuitable amino acid which results in a protein having reduced proteaseactivity. For example, one of more of E27, D43, D83, D85, D97, S99, H101E111, E136, E193, D206, D208, D210, 11212 G216, K217, R218 & E250 may bemutated to alanine. Examples of suitable mutations are E27A, D43A, D83A,D85A, D97A, S99A, H101A E111A, E136A, E193A, D206A, D208A, D210A, H212A,H212N, H212Q, G216K, K217G, K217T, R218L & E250A. In one embodiment themutated OmpT gene comprises D210A and H212A mutations. A suitablemutated OmpT sequence comprising D210A and H212A mutations is shown inSEQ ID NO: 23.

The expression “knockout mutated OmpT gene” in the context of thepresent invention means that the gene comprises one or more mutationsthereby causing no expression of the protein encoded by the gene toprovide a cell deficient in the protein encoded by the knockout mutatedgene. The knockout gene may be partially or completely transcribed butnot translated into the encoded protein. The knockout mutated OmpT genemay be mutated in any suitable way, for example by one or more deletion,insertion, point, missense, nonsense and frameshift mutations, to causeno expression of the protein. For example, the gene may be knocked outby insertion of a foreign DNA sequence, such as an antibiotic resistancemarker, into the gene coding sequence.

In one embodiment the OmpT gene comprises a mutation to the gene startcodon and/or one or more stop codons positioned downstream of the genestart codon and upstream of the gene stop codon thereby preventingexpression of the OmpT protein. The mutation to the start codon may be amissense mutation of one, two or all three of the nucleotides of thestart codon. Alternatively or additionally the start codon may bemutated by an insertion or deletion frameshift mutation.

A suitable mutated knockout OmpT sequence is shown in SEQ ID NO: 24.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated ompT gene, such asbeing deficient in chromosomal ompT.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated degP gene, such asbeing deficient in chromosomal degP. In one embodiment the gram-negativebacterial cell according to the present invention does not carry amutated degP gene.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated ptr gene, such asbeing deficient in chromosomal ptr.

Many genetically engineered mutations including knockout mutationsinvolve the use of antibiotic resistance markers which allow theselection and identification of successfully mutated cells. However,there are a number of disadvantages to using antibiotic resistancemarkers.

In one embodiment of the present invention, the mutated genes maycomprise one or more restriction marker site. Therefore, the spr geneand/or a mutated DegP gene encoding a DegP protein having chaperoneactivity but not protease activity and/or a mutated ptr gene and/or amutated OmpT gene may be mutated to comprise one or more restrictionmarker sites. The restriction sites are genetically engineered into thegene and are non-naturally occurring. The restriction marker sites areadvantageous because they allow screening and identification ofcorrectly modified cells which comprise the required chromosomalmutations. Cells which have been modified to carry one or more of themutated genes may be analyzed by PCR of genomic DNA from cell lysatesusing oligonucleotide pairs designed to amplify a region of the genomicDNA comprising a non-naturally occurring restriction marker site. Theamplified DNA may then be analyzed by agarose gel electrophoresis beforeand after incubation with a suitable restriction enzyme capable ofdigesting the DNA at the non-naturally occurring restriction markersite. The presence of DNA fragments after incubation with therestriction enzyme confirms that the cells have been successfullymodified to carry the one or more mutated genes.

In the embodiment wherein the cell comprises a knockout mutated ptr genehaving the DNA sequence of SEQ ID NO: 6, the oligonucleotide primersequences shown in SEQ ID NO: 17 and SEQ ID NO:18 may be used to amplifythe region of the DNA comprising the non-naturally occurring Ase Irestriction site from the genomic DNA of transformed cells. Theamplified genomic DNA may then be incubated with Ase I restrictionenzyme and analyzed by gel electrophoresis to confirm the presence ofthe mutated ptr gene in the genomic DNA.

In the embodiment wherein the cell comprises a mutated DegP gene havingthe DNA sequence of SEQ ID NO: 9, the oligonucleotide primer sequencesshown in SEQ ID NO: 19 and SEQ ID NO:20 may be used to amplify theregion of the DNA comprising the non-naturally occurring Ase Irestriction site from the genomic DNA of transformed cells. Theamplified genomic DNA may then be incubated with Ase I restrictionenzyme and analyzed by gel electrophoresis to confirm the presence ofthe mutated DegP gene in the genomic DNA.

The one or more restriction sites may be introduced by any suitablemutation including by one or more deletion, insertion, point, missense,nonsense and frameshift mutations. A restriction site may be introducedby the mutation of the start codon and/or mutation to introduce the oneor more stop codons, as described above. This embodiment is advantageousbecause the restriction marker site is a direct and unique marker of theknockout mutations introduced.

A restriction maker site may be inserted which comprises an in-framestop codon, such as an Ase I restriction site. This is particularlyadvantageous because the inserted restriction site serves as both arestriction marker site and a stop codon to prevent full transcriptionof the gene coding sequence. For example, in the embodiment wherein astop codon is introduced to the ptr gene by introduction of an Ase Isite, this also creates a restriction site, as shown in FIG. 9 a.

A restriction marker site may be inserted by the mutation to the startcodon and optionally one or more further point mutations. In thisembodiment the restriction marker site is preferably an EcoR Irestriction site. This is particularly advantageous because the mutationto the start codon also creates a restriction marker site. For example,in the embodiment wherein the start codon of the ptr gene is changed toATT, this creates an EcoR I marker site, as shown in FIG. 9 a.

In the embodiment of the present invention wherein the cell carries amutated OmpT gene, the one or more restriction sites may be introducedby any suitable mutation including by one or more deletion, insertion,point, missense, nonsense and frameshift mutations. For example, in theembodiment wherein the OmpT gene comprises the mutations D210A andH212A, these mutations introduce silent HindIII restriction site whichmay be used as a selection marker.

In the mutated spr gene and the mutated DegP gene, a marker restrictionsite may be introduced using silent codon changes. For example, an Ase Isite may be used as a silent restriction marker site, wherein the TAAstop codon is out-of-frame, as shown in FIG. 9 c for DegP.

In the embodiments of the present invention, wherein the ptr gene ismutated to encode a Protease III having reduced protease activity, oneor more marker restriction site may be introduced using silent codonchanges.

The recombinant gram-negative bacterial cell according to the presentinvention may be produced by any suitable means.

The skilled person knows of suitable techniques which may be used toreplace a chromosomal gene sequence with a mutated gene sequence inorder to introduce the spr mutant gene. Suitable vectors may be employedwhich allow integration into the host chromosome by homologousrecombination.

Suitable gene replacement methods are described, for example, inHamilton et al (New Method for Generating Deletions and GeneReplacements in Escherichia coli, Hamilton C. M. et al., Journal ofBacteriology September 1989, Vol. 171, No. 9 p 4617-4622), Skorupski etal (Positive selection vectors for allelic exchange, Skorupski K andTaylor R. K., Gene, 1996, 169, 47-52), Kiel et al (A general method forthe construction of Escherichia coli mutants by homologous recombinationand plasmid segregation, Kiel J. A. K. W. et al, Mol Gen Genet. 1987,207:294-301), Blomfield et al (Allelic exchange in Escherichia coliusing the Bacillus subtilis sacB gene and a temperature sensitive pSC101replicon, Blomfield I. C. et al., Molecular Microbiology 1991, 5(6),1447-1457) and Ried et al. (An nptI-sacB-sacR cartridge for constructingdirected, unmarked mutations in Gram-negative bacteria by markerexchange-eviction mutagenesis, Ried J. L. and Collmer A., Gene 57 (1987)239-246). A suitable plasmid which enables homologousrecombination/replacement is the pKO3 plasmid (Link et al., 1997,Journal of Bacteriology, 179, 6228-6237).

In the embodiment wherein the cell comprises a recombinantpolynucleotide encoding DsbC, the skilled person knows suitabletechniques which may be used to insert the recombinant polynucleotideencoding DsbC. The recombinant polynucleotide encoding DsbC may beintegrated into the cell's genome using a suitable vector such as thepKO3 plasmid.

In the embodiment wherein the cell comprises a recombinantpolynucleotide encoding a protein of interest, the skilled person alsoknows suitable techniques which may be used to insert the recombinantpolynucleotide encoding the protein of interest. The recombinantpolynucleotide encoding the protein of interest may be integrated intothe cell's genome using a suitable vector such as the pKO3 plasmid.

Alternatively or additionally, the recombinant polynucleotide encodingDsbC and/or the recombinant polynucleotide encoding a protein ofinterest may be non-integrated in a recombinant expression cassette. Inone embodiment an expression cassette is employed in the presentinvention to carry the polynucleotide encoding the DsbC and/or theprotein of interest and one or more regulatory expression sequences. Theone or more regulatory expression sequences may include a promoter. Theone or more regulatory expression sequences may also include a 3′untranslated region such as a termination sequence. Suitable promotersare discussed in more detail below.

In one embodiment an expression cassette is employed in the presentinvention to carry the polynucleotide encoding the protein of interestand/or the recombinant polynucleotide encoding DsbC. An expressioncassette typically comprises one or more regulatory expressionsequences, one or more coding sequences encoding one or more proteins ofinterest and/or a coding sequence encoding DsbC. The one or moreregulatory expression sequences may include a promoter. The one or moreregulatory expression sequences may also include a 3′ untranslatedregion such as a termination sequence. Suitable promoters are discussedin more detail below.

In one embodiment, the cell according to the present invention comprisesone or more vectors, such as plasmid. The vector preferably comprisesone or more of the expression cassettes as defined above. In oneembodiment the polynucleotide sequence encoding a protein of interestand the polynucleotide encoding DsbC are inserted into one vector.Alternatively the polynucleotide sequence encoding a protein of interestand the polynucleotide encoding DsbC are inserted into separate vectors.

In the embodiment where the protein of interest is an antibodycomprising both heavy and light chains, the cell line may be transfectedwith two vectors, a first vector encoding a light chain polypeptide anda second vector encoding a heavy chain polypeptide. Alternatively, asingle vector may be used, the vector including sequences encoding lightchain and heavy chain polypeptides. Alternatively, the polynucleotidesequence encoding the antibody and the polynucleotide encoding DsbC areinserted into one vector. Preferably the vector comprises the sequencesencoding the light and heavy chain polypeptides of the antibody.

In the embodiment wherein the cell also expresses one or more furtherproteins as follows:

-   -   one or more proteins capable of facilitating protein folding,        such as FkpA, Skp, SurA, PPiA and PPiD; and/or    -   one or more protein capable of facilitating protein secretion or        translocation, such as SecY, SecE, SecG, SecYEG, SecA, SecB,        FtsY and Lep; and/or    -   one or more proteins capable of facilitating disulphide bond        formation, such as DsbA, DsbB, DsbD, DsbG;    -   the one or more further protein may be expressed from one or        more polynucleotides inserted into the same vector as the        polynucleotide encoding DsbC and/or the polynucleotide sequence        encoding a protein of interest. Alternatively the one or more        polynucleotides may be inserted into separate vectors.

The vector for use in the present invention may be produced by insertingone or more expression cassettes as defined above into a suitablevector. Alternatively, the regulatory expression sequences for directingexpression of the polynucleotide sequence may be contained in the vectorand thus only the encoding region of the polynucleotide may be requiredto complete the vector.

The polynucleotide encoding DsbC and/or the polynucleotide encoding theprotein of interest is suitably inserted into a replicable vector,typically an autonomously replicating vector, for expression in the cellunder the control of a suitable promoter for the cell. Many vectors areknown in the art for this purpose and the selection of the appropriatevector may depend on the size of the nucleic acid and the particularcell type.

Examples of vectors which may be employed to transform the host cellwith a polynucleotide according to the invention include:

-   -   a plasmid, such as pBR322 or pACYC184, and/or    -   a viral vector such as bacterial phage    -   a transposable genetic element such as a transposon

Such vectors usually comprise a plasmid origin of DNA replication, anantibiotic selectable marker, a promoter and transcriptional terminatorseparated by a multi-cloning site (expression cassette) and a DNAsequence encoding a ribosome binding site.

The promoters employed in the present invention can be linked to therelevant polynucleotide directly or alternatively be located in anappropriate position, for example in a vector such that when therelevant polypeptide is inserted the relevant promoter can act on thesame. In one embodiment the promoter is located before the encodingportion of the polynucleotide on which it acts, for example a relevantpromoter before each encoding portion of polynucleotide. “Before” asused herein is intended to imply that the promoter is located at the 5prime end in relation to the encoding polynucleotide portion.

The promoters may be endogenous or exogenous to the host cells. Suitablepromoters include Lac, tac, trp, PhoA, Ipp, Arab, Tet and T7.

One or more promoters employed may be inducible promoters.

In the embodiment wherein the polynucleotide encoding DsbC and thepolynucleotide encoding the protein of interest are inserted into onevector, the nucleotide sequences encoding DsbC and the protein ofinterest may be under the control of a single promoter or separatepromoters. In the embodiment wherein the nucleotide sequences encodingDsbC and the protein of interest are under the control of separatepromoters, the promoter may be independently inducible promoters.

Expression units for use in bacterial systems also generally contain aShine-Dalgamo (S.D.) sequence operably linked to the DNA encoding thepolypeptide of interest. The promoter can be removed from the bacterialsource DNA by restriction enzyme digestion and inserted into the vectorcontaining the desired DNA.

In the embodiments of the present invention wherein a polynucleotidesequence comprises two or more encoding sequences for two or moreproteins of interest, for example an antibody light chain and antibodyheavy chain, the polynucleotide sequence may comprise one or moreinternal ribosome entry site (IRES) sequences which allows translationinitiation in the middle of an mRNA. An IRES sequence may be positionedbetween encoding polynucleotide sequences to enhance separatetranslation of the mRNA to produce the encoded polypeptide sequences.

The expression vector preferably also comprises a dicistronic messagefor producing the antibody or antigen binding fragment thereof asdescribed in WO 03/048208 or WO2007/039714 (the contents of which areincorporated herein by reference). Preferably the upstream cistroncontains DNA coding for the light chain of the antibody and thedownstream cistron contains DNA coding for the corresponding heavychain, and the dicistronic intergenic sequence (IGS) preferablycomprises a sequence selected from IGS1 (SEQ ID NO: 36), IGS2 (SEQ IDNO: 37), IGS3 (SEQ ID NO: 38) and IGS4 (SEQ ID NO: 39).

The terminators may be endogenous or exogenous to the host cells. Asuitable terminator is rrnB.

Further suitable transcriptional regulators including promoters andterminators and protein targeting methods may be found in “Strategiesfor Achieving High-Level Expression of Genes in Escherichia coli” SavvasC. Makrides, Microbiological Reviews, September 1996, p 512-538.

The DsbC polynucleotide inserted into the expression vector preferablycomprises the nucleic acid encoding the DsbC signal sequence and theDsbC coding sequence. The vector preferably contains a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells, preferably to replicate independently of the hostchromosome. Such sequences are well known for a variety of bacteria.

In one embodiment the DsbC and/or the protein of interest comprises ahistidine-tag at the N-terminus and/or C-terminus.

The antibody molecule may be secreted from the cell or targeted to theperiplasm by suitable signal sequences. Alternatively, the antibodymolecules may accumulate within the cell's cytoplasm. Preferably theantibody molecule is targeted to the periplasm.

The polynucleotide encoding the protein of interest may be expressed asa fusion with another polypeptide, preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature polypeptide. The heterologous signal sequence selected should beone that is recognized and processed by the host cell. For prokaryotichost cells that do not recognize and process the native or a eukaryoticpolypeptide signal sequence, the signal sequence is substituted by aprokaryotic signal sequence. Suitable signal sequences include OmpA,PhoA, LamB, Pe1B, DsbA and DsbC.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

In a preferred embodiment of the present invention the present inventionprovides a multi-cistronic vector comprising the polynucleotide sequenceencoding DsbC and the polynucleotide sequence encoding a protein ofinterest. The multicistronic vector may be produced by an advantageouscloning method which allows repeated sequential cloning ofpolynucleotide sequences into a vector. The method uses compatiblecohesive ends of a pair of restrictions sites, such as the “AT” ends ofAse I and Nde I restriction sites. A polynucleotide sequence comprisinga coding sequence and having compatible cohesive ends, such as aAseI-NdeI fragment, may be cloned into a restrictions site in thevector, such as Nde I. The insertion of the polynucleotide sequencedestroys the 5′ restriction site but creates a new 3′ restriction site,such as NdeI, which may then be used to insert a further polynucleotidesequence comprising compatible cohesive ends. The process may then berepeated to insert further sequences. Each polynucleotide sequenceinserted into the vector comprises non-coding sequence 3′ to the stopcodon which may comprise an Ssp I site for screening, a Shine Dalgarnoribosome binding sequence, an A rich spacer and an NdeI site encoding astart codon.

A diagrammatic representation of the creation of a vector comprising apolynucleotide sequence encoding a light chain of an antibody (LC), aheavy chain of an antibody (HC), a DsbC polynucleotide sequence and afurther polynucleotide sequence is shown in FIG. 10.

Successfully mutated strains may be identified using methods well knownin the art including colony PCR DNA sequencing and colony PCRrestriction enzyme mapping.

In the embodiment wherein the cell comprises two or more the mutatedgenes, the mutated protease may be introduced into the gram-negativebacterium on the same or different vectors.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated ompT gene, such asbeing deficient in chromosomal ompT.

The cell according to the present invention may further comprise apolynucleotide sequence encoding a protein of interest. Thepolynucleotide sequence encoding the protein of interest may beexogenous or endogenous. The polynucleotide sequence encoding theprotein of interest may be integrated into the host's chromosome or maybe non-integrated in a vector, typically a plasmid.

In one embodiment the cell according to the present invention expressesa protein of interest. “Protein of interest” in the context of thepresent specification is intended to refer to polypeptide forexpression, usually a recombinant polypeptide. However, the protein ofinterest may be an endogenous protein expressed from an endogenous genein the host cell.

As used herein, a “recombinant polypeptide” refers to a protein that isconstructed or produced using recombinant DNA technology. The protein ofinterest may be an exogenous sequence identical to the endogenousprotein or a mutated version thereof, for example with attenuatedbiological activity, or fragment thereof, expressed from an exogenousvector. Alternatively, the protein of interest may be a heterologousprotein, not normally expressed by the host cell.

The protein of interest may be any suitable protein including atherapeutic, prophylactic or diagnostic protein.

In one embodiment the protein of interest is useful in the treatment ofdiseases or disorders including inflammatory diseases and disorders,immune disease and disorders, fibrotic disorders and cancers.

The term “inflammatory disease” or “disorder” and “immune disease ordisorder” includes rheumatoid arthritis, psoriatic arthritis, still'sdisease, Muckle Wells disease, psoriasis, Crohn's disease, ulcerativecolitis, SLE (Systemic Lupus Erythematosus), asthma, allergic rhinitis,atopic dermatitis, multiple sclerosis, vasculitis, Type I diabetesmellitus, transplantation and graft-versus-host disease.

The term “fibrotic disorder” includes idiopathic pulmonary fibrosis(IPF), systemic sclerosis (or scleroderma), kidney fibrosis, diabeticnephropathy, IgA nephropathy, hypertension, end-stage renal disease,peritoneal fibrosis (continuous ambulatory peritoneal dialysis), livercirrhosis, age-related macular degeneration (ARMD), retinopathy, cardiacreactive fibrosis, scarring, keloids, burns, skin ulcers, angioplasty,coronary bypass surgery, arthroplasty and cataract surgery.

The term “cancer” includes a malignant new growth that arises fromepithelium, found in skin or, more commonly, the lining of body organs,for example: breast, ovary, prostate, lung, kidney, pancreas, stomach,bladder or bowel. Cancers tend to infiltrate into adjacent tissue andspread (metastasise) to distant organs, for example: to bone, liver,lung or the brain.

The protein may be a proteolytically-sensitive polypeptide, i.e.proteins that are prone to be cleaved, susceptible to cleavage, orcleaved by one or more gram-negative bacterial, such as E. coli,proteases, either in the native state or during secretion. In oneembodiment the protein of interest is proteolytically-sensitive to aprotease selected from DegP, Protease III and Tsp. In one embodiment theprotein of interest is proteolytically-sensitive to the protease Tsp. Inone embodiment the protein of interest is proteolytically-sensitive tothe proteases DegP and Protease III. In one embodiment the protein ofinterest is proteolytically sensitive to the proteases DegP and Tsp. Inone embodiment the protein of interest is proteolytically-sensitive tothe proteases Tsp and Protease III. In one embodiment the protein ofinterest is proteolytically sensitive to the proteases DegP, ProteaseIII and Tsp.

Preferably the protein is a eukaryotic polypeptide.

The protein of interest expressed by the cells according to theinvention may, for example be an immunogen, a fusion protein comprisingtwo heterologous proteins or an antibody. Antibodies for use as theprotein of interest include monoclonal, multi-valent, multi-specific,humanized, fully human or chimeric antibodies. The antibody can be fromany species but is preferably derived from a monoclonal antibody, ahuman antibody, or a humanized fragment. The antibody can be derivedfrom any class (e.g. IgG, IgE, IgM, IgD or IgA) or subclass ofimmunoglobulin molecule and may be obtained from any species includingfor example mouse, rat, shark, rabbit, pig, hamster, camel, llama, goator human. Parts of the antibody fragment may be obtained from more thanone species for example the antibody fragments may be chimeric. In oneexample the constant regions are from one species and the variableregions from another. The antibody may be a complete antibody moleculehaving full length heavy and light chains or a fragment thereof, e.g.VH, VL, VHH, Fab, modified Fab, Fab′, F(ab′)₂, Fv, scFv fragment,Fab-Fv, or a dual specificity antibody, such as a Fab-dAb, as describedin PCT/GB2008/003331.

The antibody may be specific for any target antigen. The antigen may bea cell-associated protein, for example a cell surface protein on cellssuch as bacterial cells, yeast cells, T-cells, endothelial cells ortumour cells, or it may be a soluble protein. Antigens of interest mayalso be any medically relevant protein such as those proteinsupregulated during disease or infection, for example receptors and/ortheir corresponding ligands. Particular examples of cell surfaceproteins include adhesion molecules, for example integrins such as f31integrins e.g. VLA-4, E-selectin, P selectin or L-selectin, CD2, CD3,CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD₂O, CD23, CD25, CD33,CD38, CD40, CD40L, CD45, CDW52, CD69, CD134 (OX40), ICOS, BCMP7, CD137,CD27L, CDCP1, CSF1 or CSF1-Receptor, DPCR1, DPCR1, dudulin2, FLJ20584,FLJ40787, HEK2, KIAA0634, KIAA0659, KIAAl246, KIAA1455, LTBP2, LTK,MAL2, MRP2, nectin-like2, NKCC1, PTK7, RAIG1, TCAM1, SC6, BCMP101,BCMP84, BCMP11, DTD, carcinoembryonic antigen (CEA), human milk fatglobulin (HMFG1 and 2), MHC Class I and MHC Class II antigens, KDR andVEGF, and where appropriate, receptors thereof.

Soluble antigens include interleukins such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-8, IL-12, IL-13, IL-14, IL-16 or IL-17, such as IL17Aand/or IL17F, viral antigens for example respiratory syncytial virus orcytomegalovirus antigens, immunoglobulins, such as IgE, interferons suchas interferon α, interferon β or interferon γ, tumour necrosis factorTNF (formerly known as tumour necrosis factor-α), tumor necrosisfactor-β, colony stimulating factors such as G-CSF or GM-CSF, andplatelet derived growth factors such as PDGF-α, and PDGF-β and whereappropriate receptors thereof. Other antigens include bacterial cellsurface antigens, bacterial toxins, viruses such as influenza, EBV,HepA, B and C, bioterrorism agents, radionuclides and heavy metals, andsnake and spider venoms and toxins.

In one embodiment, the antibody may be used to functionally alter theactivity of the antigen of interest. For example, the antibody mayneutralize, antagonize or agonise the activity of said antigen, directlyor indirectly.

In one aspect of the present invention there is provided a recombinantgram-negative bacterial cell comprising a mutant spr gene encoding amutant spr protein, a wild-type Tsp gene and a polynucleotide sequenceencoding an antibody or an antigen binding fragment thereof specific forTNF. The wild-type chromosomal Tsp gene is preferably a non-recombinantchromosomal Tsp gene. Preferably, the cell further comprises arecombinant polynucleotide encoding DsbC.

In a preferred embodiment the protein of interest expressed by the cellsaccording to the present invention is an anti-TNF antibody, morepreferably an anti-TNF Fab′, as described in W001/094585 (the contentsof which are incorporated herein by reference).

In a one embodiment the antibody having specificity for human TNFα,comprises a heavy chain wherein the variable domain comprises a CDRhaving the sequence shown in SEQ ID NO:28 for CDRH1, the sequence shownin SEQ ID NO:29 or SEQ ID NO:34 for CDRH2 or the sequence shown in SEQID NO:30 for CDRH3.

In one embodiment the antibody comprises a light chain wherein thevariable domain comprises a CDR having the sequence shown in SEQ IDNO:31 for CDRL1, the sequence shown in SEQ ID NO:32 for CDRL2 or thesequence shown in SEQ ID NO:33 for CDRL3.

The CDRs given in SEQ IDS NOS:28 and 30 to 34 referred to above arederived from a mouse monoclonal antibody hTNF40. However, SEQ ID NO:29consists of a hybrid CDR. The hybrid CDR comprises part of heavy chainCDR2 from mouse monoclonal antibody hTNF40 (SEQ ID NO:34) and part ofheavy chain CDR2 from a human group 3 germline V region sequence.

In one embodiment the antibody comprises a heavy chain wherein thevariable domain comprises a CDR having the sequence shown in SEQ IDNO:28 for CDRH1, the sequence shown in SEQ ID NO:29 or SEQ ID NO:34 forCDRH2 or the sequence shown in SEQ ID NO:30 for CDRH3 and a light chainwherein the variable domain comprises a CDR having the sequence shown inSEQ ID NO:31 for CDRL1, the sequence shown in SEQ ID NO:32 for CDRL2 orthe sequence shown in SEQ ID NO:33 for CDRL3.

In one embodiment the antibody comprises SEQ ID NO:28 for CDRH1, SEQ IDNO: 29 or SEQ ID NO:34 for CDRH2, SEQ ID NO:30 for CDRH3, SEQ ID NO:31for CDRL1, SEQ ID NO:32 for CDRL2 and SEQ ID NO:33 for CDRL3. Preferablythe antibody comprises SEQ ID NO:29 for CDRH2.

The anti-TNF antibody is preferably a CDR-grafted antibody molecule. Ina preferred embodiment the variable domain comprises human acceptorframework regions and non-human donor CDRs.

Preferably the antibody molecule has specificity for human TNF (formerlyknown as TNFα), wherein the light chain comprises the light chainvariable region of SEQ ID NO: 11 and the heavy chain comprises the heavychain variable region of SEQ ID NO: 12.

The anti-TNF antibody is preferably a Fab or Fab′ fragment.

Preferably the antibody molecule having specificity for human TNF is aFab′ and has a light chain sequence comprising or consisting of SEQ IDNO: 13 and a heavy chain sequence comprising or consisting of SEQ ID NO:14.

After expression, antibody fragments may be further processed, forexample by conjugation to another entity such as an effector molecule.

The term effector molecule as used herein includes, for example,antineoplastic agents, drugs, toxins (such as enzymatically activetoxins of bacterial or plant origin and fragments thereof e.g. ricin andfragments thereof) biologically active proteins, for example enzymes,other antibody or antibody fragments, synthetic or naturally occurringpolymers, nucleic acids and fragments thereof e.g. DNA, RNA andfragments thereof, radionuclides, particularly radioiodide,radioisotopes, chelated metals, nanoparticles and reporter groups suchas fluorescent compounds or compounds which may be detected by NMR orESR spectroscopy. Effector molecular may be attached to the antibody orfragment thereof by any suitable method, for example an antibodyfragment may be modified to attach at least one effector molecule asdescribed in WO05/003171 or WO05/003170 (the contents of which areincorporated herein by reference). WO05/003171 or WO05/003170 alsodescribe suitable effector molecules.

In one embodiment the antibody or fragment thereof, such as a Fab, isPEGylated to generate a product with the required properties, forexample similar to the whole antibodies, if required. For example, theantibody may be a PEGylated anti-TNF-α Fab′, as described inWO01/094585, preferably having attached to one of the cysteine residuesat the C-terminal end of the heavy chain a lysyl-maleimide-derived groupwherein each of the two amino groups of the lysyl residue has covalentlylinked to it a methoxypoly(ethyleneglycol) residue having a molecularweight of about 20,000 Da, such that the total average molecular weightof the methoxypoly(ethyleneglycol) residues is about 40,000Da, morepreferably the lysyl-maleimide-derived group is[1-[[[2-[[3-(2,5-dioxo-1-pyrrolidinyl)-1-oxopropyl]amino]ethyl]amino]-carbonyl]-1,5-pentanediyl]bis(iminocarbonyl).

The cell may also comprise further polynucleotide sequences encoding oneor more further proteins of interest.

In one embodiment one or more E. coli host proteins that in the wildtype are known to co-purify with the recombinant protein of interestduring purification are selected for genetic modification, as describedin Humphreys et al. “Engineering of Escherichia coli to improve thepurification of periplasmic Fab′ fragments: changing the pI of thechromosomally encoded PhoS/PstS protein”, Protein Expression andPurification 37 (2004) 109-118 and WO04/035792 (the contents of whichare incorporated herein by reference). The use of such modified hostproteins improves the purification process for proteins of interest,especially antibodies, produced in E. coli by altering the physicalproperties of selected E. coli proteins so they no longer co-purify withthe recombinant antibody. Preferably the E. coli protein that is alteredis selected from one or more of Phosphate binding protein (PhoS/PstS),Dipeptide binding protein (DppA), Maltose binding protein (MBP) andThioredoxin.

In one embodiment a physical property of a contaminating host protein isaltered by the addition of an amino acid tag to the C-terminus orN-terminus. In a preferred embodiment the physical property that isaltered is the isoelectric point and the amino acid tag is apoly-aspartic acid tag attached to the C-terminus. In one embodiment theE. coli proteins altered by the addition of said tag are Dipeptidebinding protein (DppA), Maltose binding protein (MBP), Thioredoxin andPhosphate binding protein (PhoS/PstS). In one specific embodiment the pIof the E. coli Phosphate binding protein (PhoS/PstS) is reduced from 7.2to 5.1 by the addition of a poly-aspartic acid tag (polyD), containing 6aspartic acid residues to the C-terminus

Also preferred is the modification of specific residues of thecontaminating E. coli protein to alter its physical properties, eitheralone or in combination with the addition of N or C terminal tags. Suchchanges can include insertions or deletions to alter the size of theprotein or amino acid substitutions to alter pI or hydrophobicity. Inone embodiment these residues are located on the surface of the protein.In a preferred embodiment surface residues of the PhoS protein arealtered in order to reduce the pI of the protein. Preferably residuesthat have been implicated to be important in phosphate binding (Bass,

U.S. Pat. No. 5,304,472) are avoided in order to maintain a functionalPhoS protein. Preferably lysine residues that project far out of thesurface of the protein or are in or near large groups of basic residuesare targeted. In one embodiment, the PhoS protein has a hexapoly-aspartic acid tag attached to the C-terminus whilst surfaceresidues at the opposite end of the molecule are targeted forsubstitution. Preferably selected lysine residues are substituted forglutamic acid or aspartic acid to confer a greater potential pI changethan when changing neutral residues to acidic ones. The designation fora substitution mutant herein consists of a letter followed by a numberfollowed by a letter. The first letter designates the amino acid in thewild-type protein. The number refers to the amino acid position wherethe amino acid substitution is being made, and the second letterdesignates the amino acid that is used to replace the wild-type aminoacid. In preferred mutations of PhoS in the present invention lysineresidues (K) 275, 107, 109, 110, 262, 265, 266, 309, 313 are substitutedfor glutamic acid (E) or glutamine (Q), as single or combined mutations,in addition lysine (K)318 may be substituted for aspartic acid (D) as asingle or combined mutation. Preferably the single mutations are K262E,K265E and K266E. Preferably the combined mutations are K265/266E andK110/265/266E. More preferably, all mutations are combined with thepolyaspartic acid (polyD) tag attached at the C-terminus and optionallyalso with the K318D substitution. In a preferred embodiment themutations result in a reduction in pI of at least 2 units. Preferablythe mutations of the present invention reduce the pI of PhoS from 7.2 tobetween about 4 and about 5.5. In one embodiment of the presentinvention the pI of the PhoS protein of E. coli is reduced from 7.2 toabout 4.9, about 4.8 and about 4.5 using the mutations polyD K318D,polyD K265/266E and polyD K110/265,1266E respectively.

The polynucleotide encoding the protein of interest may be expressed asa fusion with another polypeptide, preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature polypeptide. The heterologous signal sequence selected should beone that is recognized and processed by the host cell. For prokaryotichost cells that do not recognize and process the native or a eukaryoticpolypeptide signal sequence, the signal sequence is substituted by aprokaryotic signal sequence. Suitable signal sequences include OmpA,PhoA, LamB, Pe1B, DsbA and DsbC.

Embodiments of the invention described herein with reference to thepolynucleotide apply equally to alternative embodiments of theinvention, for example vectors, expression cassettes and/or host cellscomprising the components employed therein, as far as the relevantaspect can be applied to same.

The present invention also provides a method for producing a recombinantprotein of interest comprising culturing a recombinant gram-negativebacterial cell as described above in a culture medium under conditionseffective to express the recombinant protein of interest and recoveringthe recombinant protein of interest from the periplasm of therecombinant gram-negative bacterial cell and/or the culture medium. Inone embodiment wherein the cell comprises a recombinant polynucleotideencoding DsbC, the cell is cultured under conditions effective toexpress the recombinant polynucleotide encoding DsbC.

The gram negative bacterial cell and protein of interest preferablyemployed in the method of the present invention are described in detailabove.

When the polynucleotide encoding the protein of interest is exogenousthe polynucleotide may be incorporated into the host cell using anysuitable means known in the art. The polynucleotide sequence encodingDsbC may also be incorporated into the host cell using any suitablemeans known in the art. Typically, the polynucleotide is incorporated aspart of an expression vector which is transformed into the cell.Accordingly, in one aspect the cell according to the present inventioncomprises an expression cassette comprising the polynucleotide encodingthe protein of interest and an expression cassette comprising thepolynucleotide encoding DsbC.

The polynucleotide sequence encoding the protein of interest and thepolynucleotide sequence encoding DsbC can be transformed into a cellusing standard techniques, for example employing rubidium chloride, PEGor electroporation.

The method according to the present invention may also employ aselection system to facilitate selection of stable cells which have beensuccessfully transformed with the polynucleotide encoding the protein ofinterest. The selection system typically employs co-transformation of apolynucleotide sequence encoding a selection marker. In one embodiment,each polynucleotide transformed into the cell further comprises apolynucleotide sequence encoding one or more selection markers.Accordingly, the transformation of the polynucleotide encoding theprotein of interest and optionally the polynucleotide encoding DsbC andthe one or more polynucleotides encoding the marker occurs together andthe selection system can be employed to select those cells which producethe desired proteins.

Cells able to express the one or more markers are able tosurvive/grow/multiply under certain artificially imposed conditions, forexample the addition of a toxin or antibiotic, because of the propertiesendowed by the polypeptide/gene or polypeptide component of theselection system incorporated therein (e.g. antibiotic resistance).Those cells that cannot express the one or more markers are not able tosurvive/grow/multiply in the artificially imposed conditions. Theartificially imposed conditions can be chosen to be more or lessvigorous, as required.

Any suitable selection system may be employed in the present invention.Typically the selection system may be based on including in the vectorone or more genes that provides resistance to a known antibiotic, forexample a tetracycline, chloramphenicol, kanamycin or ampicillinresistance gene. Cells that grow in the presence of a relevantantibiotic can be selected as they express both the gene that givesresistance to the antibiotic and the desired protein.

An inducible expression system or a constitutive promoter may be used inthe present invention to express the protein of interest and/or theDsbC. In one embodiment, the expression of the polynucleotide sequenceencoding a protein of interest and the recombinant polynucleotideencoding DsbC is induced by adding an inducer to the culture medium.Suitable inducible expression systems and constitutive promoters arewell known in the art.

Any suitable medium may be used to culture the transformed cell. Themedium may be adapted for a specific selection system, for example themedium may comprise an antibiotic, to allow only those cells which havebeen successfully transformed to grow in the medium.

The cells obtained from the medium may be subjected to further screeningand/or purification as required. The method may further comprise one ormore steps to extract and purify the protein of interest as required.

The polypeptide may be recovered from the strain, including from thecytoplasm, periplasm and/or supernatant.

The specific method (s) used to purify a protein depends on the type ofprotein. Suitable methods include fractionation on immuno-affnity orion-exchange columns; ethanol precipitation; reversed-phase HPLC;hydrophobic-interaction chromatography; chromatography on silica;chromatography on an ion-exchange resin such as S-SEPHAROSE and DEAE;chromatofocusing; ammonium-sulfate precipitation; and gel filtration.

In one embodiment the method further comprises separating therecombinant protein of interest from DsbC.

Antibodies may be suitably separated from the culture medium and/orcytoplasm extract and/or periplasm extract by conventional antibodypurification procedures such as, for example, protein A-Sepharose,protein G chromatography, protein L chromatograpy, thiophilic, mixedmode resins, His-tag, FLAGTag, hydroxylapatite chromatography, gelelectrophoresis, dialysis, affinity chromatography, Ammonium sulphate,ethanol or PEG fractionation/precipitation, ion exchange membranes,expanded bed adsorption chromatography (EBA) or simulated moving bedchromatography.

The method may also include a further step of measuring the quantity ofexpression of the protein of interest and selecting cells having highexpression levels of the protein of interest.

The method may also including one or more further downstream processingsteps such as PEGylation of the protein of interest, such as an antibodyor antibody fragment.

One or more method steps described herein may be performed incombination in a suitable container such as a bioreactor.

EXAMPLES Example 1 Generation Cell Strain MXE001 (ΔTsp)

The MXE001 strain was generated as follows:

The Tsp cassette was moved as Sal I, Not I restriction fragments intosimilarly restricted pKO3 plasmids. The pKO3 plasmid uses thetemperature sensitive mutant of the pSC101 origin of replication (RepA)along with a chloramphenicol marker to force and select for chromosomalintegration events. The sacB gene which encodes for levansucrase islethal to E. coli grown on sucrose and hence (along with thechloramphenicol marker and pSC101 origin) is used to force and selectfor de-integration and plasmid curing events. This methodology had beendescribed previously (Hamilton et al., 1989, Journal of Bacteriology,171, 4617-4622 and Blomfield et al., 1991, Molecular Microbiology, 5,1447-1457). The pKO3 system removes all selective markers from the hostgenome except for the inserted gene.

The following plasmids were constructed.

pMXE191 comprising the knockout mutated Tsp gene as shown in the SEQ IDNO: 3 comprising EcoR I and Ase I restriction markers.

The plasmid was then transformed into electro-competent competent E.coli W3110 cells prepared using the method found in Miller, E. M. andNickoloff, J. A., “Escherichia coli electrotransformation,” in Methodsin Molecular Biology, vol. 47, Nickoloff, J. A. (ed.), Humana Press,Totowa, N.J., 105 (1995).

Day 1 40 μl of E. coli cells were mixed with (10 pg) 1 μl of pKO3 DNA ina chilled BioRad 0.2 cm electroporation cuvette before electroporationat 2500V, 25 μF and 2000. 1000 μl of 2×PY was added immediately, thecells recovered by shaking at 250 rpm in an incubator at 30° C. for 1hour. Cells were serially 1/10 diluted in 2×PY before 100 μl aliquotswere plated out onto 2×PY agar plates containing chloramphenicol at 20μg/ml prewarmed at 30° C. and 43° C. Plates were incubated overnight at30° C. and 43° C.

Day 2 The number of colonies grown at 30° C. gave an estimate of theefficiency of electroporation whilst colonies that survive growth at 43°C. represent potential integration events. Single colonies from the 43°C. plate were picked and resuspended in 10 ml of 2×PY. 100 μl of thiswas plated out onto 2×PY agar plates containing 5% (w/v) sucrosepre-warmed to 30° C. to generate single colonies. Plates were incubatedovernight at 30° C.

Day 3 Colonies here represent potential simultaneous de-integration andplasmid curing events. If the de-integration and curing events happenedearly on in the growth, then the bulk of the colony mass will be clonal.Single colonies were picked and replica plated onto 2×PY agar thatcontained either chloramphenicol at 20 μg/ml or 5% (w/v) sucrose. Plateswere incubated overnight at 30° C.

Day 4 Colonies that both grow on sucrose and die on chloramphenicolrepresent potential chromosomal replacement and plasmid curing events.These were picked and screened by PCR with a mutation specificoligonucleotide. Colonies that generated a positive PCR band of thecorrect size were struck out to produce single colonies on 2×PY agarcontaining 5% (w/v) sucrose and the plates were incubated overnight at30° C.

Day 5 Single colonies of PCR positive, chloramphenicol sensitive andsucrose resistant E. coli were used to make glycerol stocks, chemicallycompetent cells and act as PCR templates for a PCR reaction with 5′ and3′ flanking oligos to generate PCR product for direct DNA sequencingusing Taq polymerase.

Cell strain MXE001 was tested to confirm successful modification ofgenomic DNA carrying the mutated Tsp gene by PCR amplification of theregion of the Tsp gene comprising a non-naturally occurring Ase Irestriction site, as shown in FIGS. 1 a, 1 b and 1 c, usingoligonucleotides primers. The amplified regions of the DNA were thenanalyzed by gel electrophoresis before and after incubation with Ase Irestriction enzyme to confirm the presence of the non-naturallyoccurring Ase I restriction site in the mutated genes. This method wascarried out as follows:

The following oligos were used to amplify, using PCR, genomic DNA fromprepared E. coli cell lysates from MXE001 and W3110:

6284 Tsp 3′ (SEQ ID NO: 15) 5′-GCATCATAATTTTCTTTTTACCTC-3′ 6283 Tsp 5'(SEQ ID NO: 16) 5′-GGGAAATGAACCTGAGCAAAACGC-3′

The lysates were prepared by heating a single colony of cells for 10minutes at 95° C. in 20 ul of 1×PCR buffer. The mixture was allowed tocool to room temperature then centrifugation at 13,200 rpm for 10minutes. The supernatant was removed and labeled as ‘cell lysate’.

Each strain was amplified using the Tsp oligos pair.

The DNA was amplified using a standard PCR procedure.

  5 ul Buffer x10 (Roche)   1 ul dNTP mix (Roche, 10 mM mix) 1.5 ul 5′oligo (5 pmol) 1.5 ul 3′ oligo (5 pmol)   2 ul Cell lysate 0.5 ul TaqDNA polymerase (Roche 5U/ul) 38.5 ul  H2O

PCR cycle.

94° C.  1 minute 94° C.  1 minute 55° C.  1 minute {close oversizebrace} repeated for 30 cycles 72° C.  1 minute 72° C. 10 minutes

Once the reactions were complete 25 ul was removed to a new microfugetube for digestion with Ase I. To the 25 ul of PCR reaction 19 ul ofH2O, 5 ul of buffer 3 (NEB), 1 ul of Ase I (NEB) was added, mixed andincubated at 37° C. for 2 hours.

To the remaining PCR reaction 5 ul of loading buffer (x6) was added and20 ul was loaded onto a 0.8% TAE 200 ml agarose gel (Invitrogen) plusEthidium Bromide (5 ul of 10 mg/ml stock) and run at 100 volts for 1hour. 10 ul of size marker (Perfect DNA marker 0.1-12 Kb, Novagen) wasloaded in the final lane.

Once the Ase I digestions were complete 10 ul of loading buffer (x6) wasadded and 20 ul was loaded onto a 0.8% TAE agarose gel (Invitrogen) plusEthidium Bromide (5 ul of 10 mg/ml stock) and run at 100 volts for 1hour. 10 ul of size marker (Perfect DNA marker 0.1-12 Kb, Novagen) wasloaded in the final lane. Both gels were visualized using UVtransluminator.

The genomic fragment amplified showed the correct sized band of 2.8 Kbfor Tsp. Following digestion with Ase I this confirmed the presence ofthe introduced Ase I sites in the Tsp deficient strain MXE001 but not inthe W3110 control.

MXE001: genomic DNA amplified using the Tsp primer set and the resultingDNA was digested with Ase Ito produce 2.2 and 0.6 Kbps bands.

W3110 PCR amplified DNA was not digested by Ase I restriction enzyme.

Example 2 Generation of spr Mutants

The spr mutations were generated and selected for using acomplementation assay.

The spr gene was mutated using the Clontech® random mutagenisisdiversity PCR kit which introduced 1 to 2 mutations per 1000 bp. Themutated spr PCR DNA was cloned into an inducible expression vector [pTTOCDP870] which expresses CDP870 Fab′ along with the spr mutant. Thisligation was then electro-transformed into an E. coli strain MXE001(ΔTsp) prepared using the method found in Miller, E. M. and Nickoloff,J. A., “Escherichia coli electrotransformation,” in Methods in MolecularBiology, vol. 47, Nickoloff, J. A. (ed.), Humana Press, Totowa, N.J.,105 (1995). The following protocol was used, 40 ul of electro competentMXE001, 2.5 ul of the ligation (100 pg of DNA) was added to a 0.2 cmelectroporation cuvette, electro-transformation was performed using asBioRad Genepulser Xcell with the following conditions, 2500V, 25 μF and200Ω. After the electro-transformation 1 ml of SOC (Invitrogen)(pre-warmed to 37° C.) was added and the cells left to recover at 37° C.for 1 hour with gentle agitation.

The cells where plated onto Hypotonic agar (5 g/L Yeast extract, 2.5 g/LTryptone, 15 g/L Agar (all Difco)) and incubated at 40° C. Cells whichformed colonies were re-plated onto HLB at 43° C. to confirm restorationof the ability to grow under low osmotic conditions at high temperatureto the MXE001 strain. Plasmid DNA was prepared from the selected clonesand sequenced to identify spr mutations.

Using this method eight single, one double mutation and two multiplemutations in the spr protein were isolated which complemented the ATspphenotype as follows:

-   -   1. V98E    -   2. D133A    -   3. V135D    -   4. V135G    -   5. G147C    -   6. S95F and Y115F    -   7. I70T    -   8. N31T, Q73R, R100G, G140C    -   9. R62c, Q99P, R144c    -   10. L108S    -   11. L136P

Example 3 Generation of Mutant E. coli Cell Strains Carrying sprMutations

The individual mutations 1 to 5 identified in Example 2 and threecatalytic triad mutations of spr (C94A, H145A, H157A) and W174R wereused to generate new strains using either the wild-type W3110 E. colistrain (genotype: F-LAM-IN (rrnD-rrnE)1 rph1 (ATCC no. 27325)) to createspr mutated strains carrying a wild-type non-recombinant chromosomal Tspgene or MXE001 (ATsp) strain from Example 1 to make combined ATsp/mutantspr strains.

The following mutant E. coli cell strains were generated using a genereplacement vector system using the pKO3 homologousrecombination/replacement plasmid (Link et al., 1997, Journal ofBacteriology, 179, 6228-6237), as described in Example 1 for thegeneration of MXE001.

TABLE 1 Mutant E. coli Cell Strain Genotype Spr Vectors MXE001 ΔTsp —MXE008 ΔTsp, spr D133A pMXE339, pK03 spr D133A (-SalI) MXE009 ΔTsp, sprH157A pMXE345, pK03 spr H157A (-SalI) MXE010 spr G147C pMXE338, pK03 sprG147C (-SalI) MXE011 spr C94A pMXE343, pK03 spr C94A (-SalI) MXE012 sprH145A pMXE344, pK03 spr H145A (-SalI) MXE013 spr W174R pMXE346, pK03 sprW174R (-SalI) MXE014 ΔTsp, spr V135D pMXE340, pK03 spr V135D (-SalI)MXE015 ΔTsp, spr V98E pMXE342, pK03 spr V98E (-SalI) MXE016 ΔTsp, sprC94A pMXE343, pK03 spr C94A (-SalI) MXE017 ΔTsp, spr H145A pMXE344, pK03spr H145A (-SalI) MXE018 ΔTsp, spr V135G pMXE341, pK03 spr V135G (-SalI)

The mutant spr integration cassettes were moved as Sal I, Not Irestriction fragments into similarly restricted pKO3 plasmids.

The plasmid uses the temperature sensitive mutant of the pSC101 originof replication (RepA) along with a chloramphenicol marker to force andselect for chromosomal integration events. The sacB gene which encodesfor levansucrase is lethal to E. coli grown on sucrose and hence (alongwith the chloramphenicol marker and pSC101 origin) is used to force andselect for de-integration and plasmid curing events. This methodologyhad been described previously (Hamilton et al., 1989, Journal ofBacteriology, 171, 4617-4622 and Blomfield et al., 1991, MolecularMicrobiology, 5, 1447-1457). The pKO3 system removes all selectivemarkers from the host genome except for the inserted gene.

The following pK03 vectors were constructed, comprising the mutated sprgenes including a silent mutation within the spr sequence which removesa SalI restriction site for clone identification.

pMXE336, pK03 spr S95F (-SalI)pMXE337, pK03 spr Y115F (-SalI)pMXE338, pK03 spr G147C (-SalI)pMXE339, pK03 spr D133A (-SalI)pMXE340, pK03 spr V135D (-SalI)pMXE341, pK03 spr V135G (-SalI)pMXE342, pK03 spr V98E (-SalI)pMXE343, pK03 spr C94A (-SalI)pMXE344, pK03 spr H145A (-SalI)pMXE345, pK03 spr H157A (-SalI)pMXE346, pK03 spr W174R (-SalI)These plasmids were then transformed into chemically competent E. coliW3110 cells prepared using the method found in Miller, E. M. andNickoloff, J. A., “Escherichia coli electrotransformation,” in Methodsin Molecular Biology, vol. 47, Nickoloff, J. A. (ed.), Humana Press,Totowa, N.J., 105 (1995) or into MXE001 strain from Example 1 to makecombined ATsp/mutant spr strains, as shown in Table 1.

Day 1 40 μl of electro-compentent E. coli cells or MXE001 cells weremixed with (10 pg) 1 μl of pKO3 DNA in a chilled BioRad 0.2 cmelectroporation cuvette before electroporation at 2500V, 25 μF and 200Ω.1000 μl of 2×PY was added immediately, the cells recovered by shaking at250 rpm in an incubator at 30° C. for 1 hour. Cells were serially 1/10diluted in 2×PY before 100 μl aliquots were plated out onto 2×PY agarplates containing chloramphenicol at 20 μg/ml prewarmed at 30° C. and43° C. Plates were incubated overnight at 30° C. and 43° C.

Day 2 The number of colonies grown at 30° C. gave an estimate of theefficiency of electroporation whilst colonies that survive growth at 43°C. represent potential integration events. Single colonies from the 43°C. plate were picked and resuspended in 10 ml of 2×PY. 100 μl of thiswas plated out onto 2×PY agar plates containing 5% (w/v) sucrosepre-warmed to 30° C. to generate single colonies. Plates were incubatedovernight at 30° C. Day 3 Colonies here represent potential simultaneousde-integration and plasmid curing events. If the de-integration andcuring events happened early on in the growth, then the bulk of thecolony mass will be clonal. Single colonies were picked and replicaplated onto 2×PY agar that contained either chloramphenicol at 20 μg/mlor 5% (w/v) sucrose. Plates were incubated overnight at 30° C.

Day 4 Colonies that both grow on sucrose and die on chloramphenicolrepresent potential chromosomal replacement and plasmid curing events.These were picked and screened by PCR plus restriction digest for theloss of a SalI site. Colonies that generated a positive PCR band of thecorrect size and resistance to digestion by SalI were struck out toproduce single colonies on 2×PY agar containing 5% (w/v) sucrose and theplates were incubated overnight at 30° C.

Day 5 Single colonies of PCR positive, chloramphenicol sensitive andsucrose resistant E. coli were used to make glycerol stocks, chemicallycompetent cells and act as PCR templates for a PCR reaction with 5′ and3′ flanking oligos to generate PCR product for direct DNA sequencingusing Taq polymerase to confirm the correct mutation.

Example 4 Generation of Plasmid for Fab′ and DsbC Co-Expression

A plasmid was constructed containing both the heavy and light chainsequences of an anti-TNF Fab′ (an anti-TNF Fab′ having a light chainsequence shown in SEQ ID NO: 13 and a heavy chain sequence shown in SEQID NO: 14) and the sequence encoding DsbC.

A dicistronic message was created of the anti-TNFα Fab′ fragment(referred to as CDP870) described in WO01/94585. The upstream cistronencoded the light chain of the antibody (SEQ ID NO: 13) whilst thedownstream cistron encoded the heavy chain of the antibody (SEQ ID NO:14). A DNA sequence encoding the OmpA signal peptide was fused to the 5′end of the DNA coding for each of the light chain and the heavy chain toallow efficient secretion to the periplasm. The intergenic sequence(IGS2) was used as shown in SEQ ID NO: 37.

Plasmid pDPH358 (pTTO 40.4 CDP870 IGS2), an expression vector for theCDP870 Fab′ (an anti-TNF Fab′) and DsbC (a periplasmic polypeptide), wasconstructed using conventional restriction cloning methodologies whichcan be found in Sambrook et al 1989, Molecular cloning: a laboratorymanual. CSHL press, N.Y. The plasmid pDPH358 contained the followingfeatures; a strong tac promoter and lac operator sequence. As shown inFIG. 10, the plasmid contained a unique EcoRI restriction site after thecoding region of the Fab′ heavy chain, followed by a non-coding sequenceand then a unique NdeI restriction site. The DsbC gene was PCR clonedusing W3110 crude chromosomal DNA as a template such that the PCRproduct encoded for a 5′ EcoRI site followed by a strong ribosomebinding, followed by the native start codon, signal sequence and maturesequence of DsbC, terminating in a C-terminal His tag and finally anon-coding NdeI site. The EcoRI-NdeI PCR fragment was restricted andligated into the expression vector such that all three polypeptides:Fab′ light chain, Fab′ heavy chain and DsbC were encoded on a singlepolycistronic mRNA.

The Fab light chain, heavy chain genes and DcbC gene were transcribed asa single polycistronic message. DNA encoding the signal peptide from theE. coli OmpA protein was fused to the 5′ end of both light and heavychain gene sequences, which directed the translocation of thepolypeptides to the E. coli periplasm. Transcription was terminatedusing a dual transcription terminator rrnB tlt2. The lacIq gene encodedthe constitutively expressed Lac I repressor protein. This repressedtranscription from the tac promoter until de-repression was induced bythe presence of allolactose or IPTG. The origin of replication used wasp15A, which maintained a low copy number. The plasmid contained atetracycline resistance gene for antibiotic selection.

Example 5 Expression of Anti-TNF Fab′ or Anti-TNF Fab′ and DsbC in theE. coli Strains Expression of Anti-TNF Fab′ and DsbC

The wild-type W3110 cell line, the MXE001 strain provided in Example 1and the mutant strain MXE012 (H145A spr mutant strain) provided inExample 3 were transformed with the plasmid generated in Example 4.

The transformation of the strains was carried out using the method foundin Chung C. T et al Transformation and storage of bacterial cells in thesame solution. PNAS 86:2172-2175 (1989).

Expression of Anti-TNF Fab′

The wild-type W3110 cell line, spr mutant strains MXE008, MXE012, MXE017and MXE012 (H145A spr mutant strain) provided in Example 3 and theMXE001 strain provided in Example 1 were transformed with plasmidpMXE117 (pTTO CDP870 or 40.4 IGS17), an expression vector for the CDP870Fab′ (an anti-TNF Fab′ having a light chain sequence shown in SEQ ID NO:13 and a heavy chain sequence shown in SEQ ID NO: 14), was constructedusing conventional restriction cloning methodologies which can be foundin Sambrook et al 1989, Molecular cloning: a laboratory manual. CSHLpress, N.Y. The plasmid pMXE117 (pTTO CDP870 or 40.4 IGS17) containedthe following features; a strong tac promoter and lac operator sequence.The Fab light and heavy chain genes were transcribed as a singledicistronic message. DNA encoding the signal peptide from the E. coliOmpA protein was fused to the 5′ end of both light and heavy chain genesequences, which directed the translocation of the polypeptides to theE. coli periplasm. Transcription was terminated using a dualtranscription terminator rrnB tlt2. The lacIq gene encoded theconstitutively expressed Lac I repressor protein. This repressedtranscription from the tac promoter until de-repression was induced bythe presence of allolactose or IPTG. The origin of replication used wasp15A, which maintained a low copy number. The plasmid contained atetracycline resistance gene for antibiotic selection.

The transformation of the strains was carried out using the method foundin Chung C. T et al Transformation and storage of bacterial cells in thesame solution. PNAS 86:2172-2175 (1989).

Example 6 Expression of an Anti-TNF Fab′ in Mutated E. coli StrainsUsing Shake Flask Cultures

The following strains as produced by Example 5 expressing anti-TNF Fab′:W3110, MXE001, MXE012 and MXE017 were tested in a shake flask experimentcomparing growth and expression of the Fab′.

The shake flask experimental protocol used was performed as follows:

5 ml Shake Flask Experiment

A single colony was picked into 5 ml LB plus tetracycline at 10 ug/mland grown overnight at 30° C. with shaking at 250 rpm.

The overnight culture was use to inoculate 100 ml plus tetracycline to0.1 OD600. (i.e. for OD of 4, 100/4x01=2.5 mls in 100 ml.)

3×5 ml culture tubes were set up for every time point required usingthis master culture. 1 reference culture was set up to sample for ODmeasurement.

The cultures were shaken at 30° C. 250 rpm monitoring growth visually atfirst, then by sampling the reference culture to catch cultures at 0.5OD600 (usually about 2 hrs). IPTG was added to each culture tube to aconcentration of 200 uM (25 ul of 0.04M) once the culture had achievedan OD greater than 0.5.

The culture tubes were removed at the required time points e.g. 1 hr, 2hr, post induction and kept on ice.

After centrifugation at 13,200 rpm for 5 minutes the cell pellet wasre-suspended in 200 ul of periplasmic extraction buffer (100 mMTris.Cl/10 mM EDTA pH 7.4). Periplasmic extracts were agitated at 250rpm over night at 30° C. The next day, the extracts were centrifuged for10 minutes at 13,200 rpm, the supernatant decanted off and stored at−20° C. as ‘periplasmic extract’. The spent cell pellet was discarded.

ELISA Quantification.

96 well ELISA plates were coated overnight at 4° C. with AB141 (rabbitanti-human CHL UCB) at 2 μgml−1 in PBS. After washing 3× with 300 ul ofsample/conjugate buffer (PBS, BSA 0.2% (w/v), Tween 20 0.1% (v/v)),serial ½ dilutions of samples and standards were performed on the platein 100 μl of sample/conjugate buffer, and the plate agitated at 250r.p.m at room temperature for 1 hour. After washing 3× with 300 ul ofwash buffer (PBS, Tween 20 0.1% (v/v)), 100 μA of the revealing antibody6062 (rabbit anti-human kappa HRP conjugated, The Binding Site,Birmingham, U.K.) was added, after dilution at 1/1000 insample/conjugate buffer. The plate was then agitated at 250 r.p.m atroom temperature for 1 hour. After washing with 3×300 ul of wash buffer,100 ul of TMB substrate was added (50:50 mix of TMB solution(Calbiochem): dH2O) and the A₆₃₀ recorded using an automated platereader. The concentration of Fab′ in the periplasmic extracts werecalculated by comparison with purified Fab′ standards of the appropriateisotype.

FIG. 1 shows the improved growth of MXE012 and MXE017 compared to thewild-type W3110 and MXE001.

FIG. 2 shows improved expression of the Fab′ in MXE012 and MXE017compared to the wild-type W3110 and MXE001.

Example 7 Growth of E. coli Strains Expressing Anti-TNF Fab′ or Anti-TNFFab′ and DsbC Using High Density Fermentations

The following strains, as produced by example 5 were tested infermentation experiments comparing growth and expression of an anti-TNFαFab′:

Strains expressing anti-TNF Fab′ produced in Example 5:

W3100

MXE012 (H145A spr mutant strain)

Strains expressing anti-TNF Fab′ and DsbC produced in Example 5:

W3110

MXE012 (H145A spr mutant strain)Growth medium.

The fermentation growth medium was based on SM6E medium (described inHumphreys et al., 2002, Protein Expression and Purification, 26,309-320) with 3.86 g/l NaH₂PO₄.H₂O and 112 g/l glycerol.

Inoculum. Inoculum cultures were grown in the same medium supplementedwith 10 μg/ml tetracycline. Cultures were incubated at 30° C. withagitation for approximately 22 hours.

Fermentation. Fermenters (2.5 litres total volume) were seeded withinoculum culture to 0.3-0.5 OD₆₀₀. Temperature was maintained at 30° C.during the growth phase and was reduced to 25° C. prior to induction.The dissolved oxygen concentration was maintained above 30% airsaturation by variable agitation and airflow. Culture pH was controlledat 7.0 by automatic titration with 15% (v/v) NH₄OH and 10% (v/v) conc.H₂SO₄. Foaming was controlled by the addition of 10% (v/v) Struktol J673solution (Schill and Seilacher). A number of additions were made atdifferent stages of the fermentation. When biomass concentration reachedapproximately 40 OD₆₀₀, magnesium salts and NaH₂PO₄.H₂O were added.Further additions of NaH₂PO₄.H₂O were made prior to and during theinduction phase to ensure phosphate was maintained in excess. When theglycerol present at the beginning of fermentation had depleted(approximately 75 M_(ao)) a continuous feed of 80% (w/w) glycerol wasapplied. At the same point in the fermentation an IPTG feed at 170 μMwas applied. The start of IPTG feeding was taken as the start ofinduction. Fermentations were typically run for 64-120 hours at glycerolfeed rates (ranging between 0.5 and 2.5 ml/h).

Measurement of biomass concentration and growth rate. Biomassconcentration was determined by measuring the optical density ofcultures at 600 nm.

Periplasmic Extraction. Cells were collected from culture samples bycentrifugation. The supernatant fraction was retained (at −20° C.) forfurther analysis. The cell pellet fraction was resuspended to theoriginal culture volume in extraction buffer (100 mM Tris-HCl, 10 mMEDTA; pH 7.4). Following incubation at 60° C. for approximately 1.6hours the extract was clarified by centrifugation and the supernatantfraction retained (at −20° C.) for analysis.

Fab′ quantification. Fab′ concentrations in periplasmic extracts andculture supernatants were determined by Fab′ assembly ELISA as describedin Humphreys et al., 2002, Protein Expression and Purification, 26,309-320 and using Protein G hplc. A HiTrap Protein-G HP 1 ml column(GE-Healthcare or equivalent) was loaded with analyte (approximatelyneutral pH, 30° C., 0.2 um filtered) at 2 ml/min, the column was washedwith 20mM phosphate, 50 mM NaCl pH 7.4 and then Fab′ eluted using aninjection of 50 mM Glycine/HCl pH 2.7. Eluted Fab′ was measured by A280on a Agilent 1100 or 1200 HPLC system and quantified by reference to astandard curve of a purified Fab′ protein of known concentration.

FIG. 3 shows the growth profile of W3110 and MXE012 expressing anti-TNFFab′ during fermentation with an extended run time. The data illustratesa small increase in initial growth rate of the spr strain relative towild type during biomass accumulation and increased duration of survivalof the spr mutant strain MXE012 relative to wild type strain W3110 inthe last ˜20 hours of the fermentation.

FIG. 4 shows periplasmic Fab′ accumulation (filled lines and symbols)and media Fab′ accumulation (dashed lines and open symbols) for W3110and MXE012 (W3110 spr H145A) expressing anti-TNF Fab′ duringfermentation with an extended run time. The data show that the initialrates of periplasmic Fab′ accumulation are very similar for the twostrains, but that the wild type W3110 cells leak periplasmic Fab′ laterin the fermentation compared to MXE012.

FIG. 5 shows the growth profile of anti-TNFα Fab′ expressing strainsW3110 and MXE012 and of anti-TNFα Fab′ and recombinant DsbC expressingstrains W3110 and MXE012. It can be seen that the strains expressingDsbC exhibit improved growth compared to the corresponding cell strainswhich do not express recombinant DsbC. It can also be seen that thepresence of the spr mutation in the strains improves cell growth.

FIG. 6 shows total Fab yield from the periplasm (shaded symbols) andsupernatant (open unshaded symbols) from anti-TNFα Fab′ expressing E.colit strains W3110 and MXE012 and from anti-TNFα Fab′ and recombinantDsbC expressing E. coli strains W3110 and MXE012. It can be seen fromthis graph that the strains expressing recombinant DsbC produced a highyield of anti-TNFα Fab′ with strain MXE012 producing over 3.0 g/L inapproximately 92 hours. It can also be seen that the MXE012 strainscarrying a mutant spr gene exhibited reduced lysis compared to the W3110strains which can be seen as less supernatant anti-TNFα Fab′ (opensymbols).

Example 8 Determination of DNA Leakage and Total Protein Quantity inStrains

dsDNA Assay

The double-stranded DNA leakage into the supernatant of strains W3110,MXE001, MXE008 and MXE012 was determined using the Quant-IT PicogreendsDNA assay kit (Invitrogen, Ref: P11496). A standard curve was preparedby diluting the DNA standard provided in the range of 1-1000 ng/mL.Samples were diluted in TE buffer, so that the fluorescence reading fellwithin the linear range of the method (500 to 1000 times). In a 96-wellplate, 100 μl, of diluted sample or standard were mixed with 100 μL ofthe Picogreen reagent, and the plate was incubated for 5 minutes at roomtemperature, protected from light. The fluorescence counts were measuredfor 0.1s using a 485 nm excitation filter, and a 535 nm emission filter.The results are shown in FIG. 7.

Protein Assay:

The total proteins concentration of strains W3110, MXE001, MXE008 andMXE012 was determined using the Coomassie Plus Bradford assay kit(Pierce, Ref: 23236). A standard curve was made by diluting Bovine SerumAlbumin standard over a range of 25-1000 μg/mL Samples were diluted inwater so that the optical density fell within the linear range of themethod (5 to 10 times), and 33 pt of sample or standard were mixed with1 mL of coomassie reagent. After incubating for 10 minutes at roomtemperature, the OD_(595nm) was read on a spectrophotometer withcoomassie reagent as a blank. The total proteins concentration wascalculated based on the standard curve. The results are shown in FIG. 8.

Example 9 Growth of E. coli Strains Expressing Anti-TNF Fab′ and DsbCUsing Large Scale Fermentations

The following strain, as produced by example 5 was tested infermentation experiments comparing growth and viability of the strainand the expression of an anti-TNFα Fab′: MXE012 (spr H145A mutant)expressing anti-TNF Fab′ and DsbC produced in Example

The fermentations were carried out as follows:

The MXE012 expressing anti-TNF Fab′ and DsbC cells were grown initiallyusing a complex medium of yeast extract and peptone in shake flaskculture. The cells were then transferred to a seed stage fermenter usinga chemically defined medium. The cells were grown under non-nutrientlimiting conditions until a defined transfer point. The cells were thentransferred to a 250 L production fermenter using a similar chemicallydefined medium with a final volume of approximately 230 L. The culturewas initially grown in batch mode until carbon source depletion. Aftercarbon source depletion a feed limiting the carbon source was fed at anexponentially increasing rate. After the addition of a defined quantityof carbon source the rate of feed solution addition was decreased andIPTG was added to induce expression of the Fab′. The fermentation wasthen continued and the Fab′ accumulated in the periplasm. At a definedperiod after induction the culture was harvested by centrifugation andthe Fab′ was extracted from the cells by resuspending the harvestedcells in a Tris and EDTA buffer and heating to 59° C.

The growth profiles of the fermentations were determined by measuringthe optical density of culture at 600 nm.

The Fab′ titres were determined by Protein G HPLC as described inExample 7 above except that during the periplasmic extraction freshcells were used and 1 mL of extraction buffer was added to the cellculture. The supernatant and periplasmic Fab′ were measured as describedin Example 7. FIG. 12 shows the periplasmic Fab′ titre.

The cell culture viability was measured by flow cytometry usingFluorescence-Activated Cell Sorting.

FIG. 11 shows the growth profiles of 200 L fermentations of anti-TNFαFab′ and recombinant DsbC expressing strain MXE012.

FIG. 12 shows the periplasmic anti-TNFα Fab′ titres of 200 Lfermentations of anti-TNFα Fab′ and recombinant DsbC expressing strainMXE012.

FIG. 13 shows the viabilities of 200 L fermentations of anti-TNFα Fab′and recombinant DsbC expressing strain MXE012.

Example 10 Growth of E. coli strains expressing anti-TNF Fab′ and DsbCusing large scale fermentations

The following strain, as produced by example 5 was tested infermentation experiments comparing growth of the strain and theexpression of an anti-TNFα Fab′:

MXE012 expressing anti-TNF Fab′ and DsbC produced in Example 5

The fermentations were carried out as described in Example 9 with a 3000L production fermenter containing a final volume of approximately 2650L.

The growth profiles of the fermentations were determined by measuringthe optical density of culture at 600 nm.

The Fab′ titres were determined by Protein G HPLC as described inExample 9 above.

FIG. 14 shows the growth profiles of 3000 L fermentations of anti-TNFαFab′ and recombinant DsbC expressing strain MXE012.

FIG. 15 shows the periplasmic anti-TNFα Fab′ titres of 3000 Lfermentations of anti-TNFα Fab′ and recombinant DsbC expressing strainMXE012.

While this invention has been particularly shown and described withreference to preferred embodiments, it will be understood to thoseskilled in the art that various changes in form and detail may be madewithout departing from the scope of the invention as defined by theappendant claims.

1-22. (canceled)
 23. A recombinant gram-negative bacterial cellcomprising a mutant spr gene encoding a mutant spr protein and whereinthe cell comprises a non-recombinant wild-type chromosomal Tsp gene. 24.The cell according to claim 23, wherein the mutant spr gene encodes aspr protein having a mutation at one or more amino acids selected fromthe group consisting of H145, N31, R62, I70, Q73, C94, S95, V98, Q99,R100, L108, Y115, D133, V135, L136, G140, R144, G147, H157 and W174. 25.The cell according to claim 24, wherein the mutant spr gene encodes anspr protein having one or more mutations selected from the groupconsisting of H145A, N31Y, R62C, 170T, Q73R, C94A, S95F, V98E, Q99P,R100G, L108S, Y115F, D133A, V135D, V135G, L136P, G140C, R144c, G147C,H157A and W174R.
 26. The cell according to claim 25, wherein the one ormore spr protein mutations are selected from the group consisting ofS95F, V98E, Y115F, D133A, V135D, V135G and G147C.
 27. The cell accordingto claim 26, wherein the mutant spr gene encodes a spr protein havingthe mutations S95F and Y115F.
 28. The cell according to claim 25,wherein the spr protein mutation is H145A.
 29. The cell according toclaim 23, wherein the cell is isogenic to a wild-type bacterial cellexcept for the mutated spr gene.
 30. The cell according to claim 23,wherein the cell further comprises a recombinant polynucleotide encodingDsbC.
 31. The cell according to claim 23, wherein the cell furthercomprises one or more of the following mutated genes: a) a mutated DegPgene encoding a DegP protein having chaperone activity and reducedprotease activity; b) a mutated ptr gene, wherein the mutated ptr geneencodes a Protease III protein having reduced protease activity or is aknockout mutated ptr gene; and c) a mutated OmpT gene, wherein themutated OmpT gene encodes a OmpT protein having reduced proteaseactivity or is a knockout mutated OmpT gene.
 32. The cell according toclaim 23, wherein the cell is E. coli.
 33. The cell according to claim23, wherein the cell comprises a polynucleotide sequence encoding aprotein of interest.
 34. The cell according to claim 33, wherein thecell comprises a vector comprising the recombinant polynucleotideencoding DsbC and the polynucleotide sequence encoding a protein ofinterest.
 35. The cell according to claim 34, wherein the vectorcomprises a promoter which controls the expression of the recombinantpolynucleotide encoding DsbC and the polynucleotide sequence encoding aprotein of interest.
 36. The cell according to claim 33, wherein theprotein of interest is an antibody or an antigen binding fragmentthereof.
 37. The cell according to claim 36, wherein the antibody orantigen binding fragment thereof is specific for TNF.
 38. A recombinantgram-negative bacterial cell comprising a mutant spr gene encoding amutant spr protein, a wild-type Tsp gene and a polynucleotide sequenceencoding an antibody or an antigen binding fragment thereof specific forTNF.
 39. The cell according to claim 38, wherein the cell comprises arecombinant polynucleotide encoding DsbC.
 40. A method for producing arecombinant protein of interest comprising culturing a recombinantgram-negative bacterial cell as defined in claim 23 in a culture mediumunder conditions effective to express the recombinant protein ofinterest and recovering the recombinant protein of interest from theperiplasm of the recombinant gram-negative bacterial cell and/or theculture medium.
 41. The method according to claim 40, wherein therecombinant protein of interest is recovered from the periplasm and/orthe supernatant.
 42. The method according to claim 40, wherein the cellcomprises a recombinant polynucleotide encoding DsbC and the cell iscultured under conditions effective to express the recombinantpolynucleotide encoding DsbC.
 43. The method according to claim 42,wherein the expression of the polynucleotide sequence encoding a proteinof interest and the recombinant polynucleotide encoding DsbC is inducedby adding an inducer to the culture medium.
 44. The method according toclaim 42, wherein the method further comprises separating therecombinant protein of interest from DsbC.